Normal Enterochromaffin Cells Research Articles

A plausible role for actin gamma smooth muscle 2 (ACTG2) in small intestinal neuroendocrine tumorigenesis.

BackgroundSmall intestinal neuroendocrine tumors (SI-NETs) originate from the enterochromaffin cells in the ileum and jejunum. The knowledge about genetic and epigenetic abnormalities is limited. Low mRNA expression levels of actin gamma smooth muscle 2 (ACTG2) have been demonstrated in metastases relative to primary SI-NETs. ACTG2 and microRNA-145 (miR-145) are aberrantly expressed in other cancers and ACTG2 can be induced by miR-145. The aim of this study was to investigate the role of ACTG2 in small intestinal neuroendocrine tumorigenesis.MethodsProtein expression was analyzed in SI-NETs (n = 24) and in enterochromaffin cells by immunohistochemistry. The cell line CNDT2.5 was treated with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep), the selective EZH2 inhibitor EPZ-6438, or 5-aza-2’-deoxycytidine, a DNA hypomethylating agent. Cells were transfected with ACTG2 expression plasmid or miR-145. Western blotting analysis, quantitative RT-PCR, colony formation- and viability assays were performed. miR-145 expression levels were measured in tumors.ResultsEight primary tumors and two lymph node metastases displayed variable levels of positive staining. Fourteen SI-NETs and normal enterochromaffin cells stained negatively. Overexpression of ACTG2 significantly inhibited CNDT2.5 cell growth. Treatment with DZNep or transfection with miR-145 induced ACTG2 expression (>10-fold), but no effects were detected after treatment with EPZ-6438 or 5-aza-2’-deoxycytidine. DZNep also induced miR-145 expression. SI-NETs expressed relatively low levels of miR-145, with reduced expression in metastases compared to primary tumors.ConclusionsACTG2 is expressed in a fraction of SI-NETs, can inhibit cell growth in vitro, and is positively regulated by miR-145. Theoretical therapeutic strategies based on these results are discussed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12902-016-0100-3) contains supplementary material, which is available to authorized users.

Chromogranin A and Its Fragments as Regulators of Small Intestinal Neuroendocrine Neoplasm Proliferation

IntroductionChromogranin A is a neuroendocrine secretory product and its loss is a feature of malignant NEN de-differentiation. We hypothesized that chromogranin A fragments were differentially expressed during NEN metastasis and played a role in the regulation of NEN proliferation.MethodsChromogranin A mRNA (PCR) and protein (ELISA/western blot) were studied in 10 normal human mucosa, 5 enterochromaffin cell preparations, 26 small intestinal NEN primaries and 9 liver metastases. Cell viability (WST-1 assay), proliferation (bromodeoxyuridine ELISA) and expression of AKT/AKT-P (CASE ELISA/western blot) in response to chromogranin A silencing, inhibition of prohormone convertase and mTOR inhibition (RAD001/AKT antisense) as well as different chromogranin A fragments were examined in 4 SI-NEN cell lines.ResultsChromogranin A mRNA and protein levels were increased (37-340 fold, p<0.0001) in small intestinal NENs compared to normal enterochromaffin cells. Western blot identified chromogranin A-associated processing bands including vasostatin in small intestinal NENs as well as up-regulated expression of prohormone convertase in metastases. Proliferation in small intestinal NEN cell lines was decreased by silencing chromogranin A as well as by inhibition of prohormone convertase (p<0.05). This inhibition also decreased secretion of chromogranin A (p<0.05) and 5-HT (p<0.05) as well as expression of vasostatin. Metastatic small intestinal NEN cell lines were stimulated (50-80%, p<0.05) and AKT phosphorylated (Ser473: p<0.05) by vasostatin I, which was completely reversed by RAD001 (p<0.01) and AKT antisense (p<0.05) while chromostatin inhibited proliferation (~50%, p<0.05).ConclusionChromogranin A was differentially regulated in primary and metastatic small intestinal NENs and cell lines. Chromogranin A fragments regulated metastatic small intestinal NEN proliferation via the AKT pathway indicating that CgA plays a far more complex role in the biology of these tumors than previously considered.

Global microRNA profiling of well-differentiated small intestinal neuroendocrine tumors

Well-differentiated small intestinal neuroendocrine tumors are rare malignancies. They arise from enterochromaffin cells and very little is known about differential microRNA (miRNA) expression. The aim of this study was to identify the miRNA profile of well-differentiated small intestinal neuroendocrine tumors, which may have a critical role in tumor development, progression and potentially develop miRNAs as novel clinical biomarkers. Specimens from two test groups, 24 small intestinal neuroendocrine tumor specimens at different stages of malignancy, are included in this study. Total RNA from the first test group, five primary tumors, five mesentery metastases and five liver metastases was hybridized onto the Affymetrix Genechip miRNA arrays to perform a genome-wide profile. The results were validated by using quantitative real-time PCR (QRT-PCR) and northern blot analyses. We then expanded the investigation to laser capture microdissected small intestinal neuroendocrine tumor cells and immuno-laser capture microdissected normal enterochromaffin cells of the first test group. Furthermore, a second test group, three primary tumors, three mesentery metastases and three liver metastases, was included in the study. Thus, two independent test groups validated the data by QRT-PCR. Moreover, we characterized nine miRNAs, five (miR-96, -182, -183, -196a and -200a), which are upregulated during tumor progression, whereas four (miR-31, -129-5p, -133a and -215) are downregulated. Several online software programs were used to predict potential miRNA target genes to map a number of putative target genes for the aberrantly regulated miRNAs, through an advanced and novel bioinformatics analysis. Our findings provide information about pivotal miRNAs, which may lead to further insights into tumorigenesis, progression mechanisms and novel therapeutic targets recognition.

The Stimulatory Adenosine Receptor ADORA2B Regulates Serotonin (5-HT) Synthesis and Release in Oxygen-Depleted EC Cells in Inflammatory Bowel Disease

ObjectiveWe recently demonstrated that hypoxia, a key feature of IBD, increases enterochromaffin (EC) cell 5-HT secretion, which is also physiologically regulated by the ADORA2B mechanoreceptor. Since hypoxia is associated with increased extracellular adenosine, we wanted to examine whether this nucleotide amplifies HIF-1α-mediated 5-HT secretion.DesignThe effects of hypoxia were studied on IBD mucosa, isolated IBD-EC cells, isolated normal EC cells and the EC cell tumor derived cell line KRJ-1. Hypoxia (0.5% O2) was compared to NECA (adenosine agonist), MRS1754 (ADORA2B receptor antagonist) and SCH442146 (ADORA2A antagonist) on HIF signaling and 5-HT secretion. Antisense approaches were used to mechanistically evaluate EC cells in vitro. PCR and western blot were used to analyze transcript and protein levels of HIF-1α signaling and neuroendocrine cell function. An animal model of colitis was evaluated to confirm hypoxia:adenosine signaling in vivo.ResultsHIF-1α is upregulated in IBD mucosa and IBD-EC cells, the majority (∼90%) of which express an activated phenotype in situ. Hypoxia stimulated 5-HT release maximally at 30 mins, an effect amplified by NECA and selectively inhibited by MRS1754, through phosphorylation of TPH-1 and activation of VMAT-1. Transient transfection with Renilla luciferase under hypoxia transcriptional response element (HRE) control identified that ADORA2B activated HIF-1α signaling under hypoxic conditions. Additional signaling pathways associated with hypoxia:adenosine included MAP kinase and CREB. Antisense approaches mechanistically confirmed that ADORA2B signaling was linked to these pathways and 5-HT release under hypoxic conditions. Hypoxia:adenosine activation which could be reversed by 5′-ASA treatment was confirmed in a TNBS-model.ConclusionHypoxia induced 5-HT synthesis and secretion is amplified by ADORA2B signaling via MAPK/CREB and TPH-1 activation. Targeting ADORA2s may decrease EC cell 5-HT production and secretion in IBD.

Differential signal pathway activation and 5-HT function: the role of gut enterochromaffin cells as oxygen sensors

The chemomechanosensory function of the gut enterochromaffin (EC) cell enables it to respond to dietary agents and mechanical stretch. We hypothesized that the EC cell, which also sensed alterations in luminal or mucosal oxygen level, was physiologically sensitive to fluctuations in O(2). Given that low oxygen levels induce 5-HT production and secretion through a hypoxia inducible factor 1α (HIF-1α)-dependent pathway, we also hypothesized that increasing O(2) would reduce 5-HT production and secretion. Isolated normal EC cells as well as the well-characterized EC cell model KRJ-I were used to examine HIF signaling (luciferase-assays), hypoxia transcriptional response element (HRE)-mediated transcription (PCR), signaling pathways (Western blot), and 5-HT release (ELISA) during exposure to different oxygen levels. Normal EC cells and KRJ-I cells express HIF-1α, and transient transfection with Renilla luciferase under HRE control identified a hypoxia-mediated pathway in these cells. PCR confirmed activation of HIF-downstream targets, GLUT1, IGF2, and VEGF under reduced O(2) levels (0.5%). Reducing O(2) also elevated 5-HT secretion (2-3.2-fold) as well as protein levels of HIF-1α (1.7-3-fold). Increasing O(2) to 100% inhibited HRE-mediated signaling, transcription, reduced 5-HT secretion, and significantly lowered HIF-1α levels (∼75% of control). NF-κB signaling was also elevated during hypoxia (1.2-1.6-fold), but no significant changes were noted in PKA/cAMP. We concluded that gut EC cells are oxygen responsive, and alterations in O(2) levels differentially activate HIF-1α and tryptophan hydroxylase 1, as well as NF-κB signaling. This results in alterations in 5-HT production and secretion and identifies that the chemomechanosensory role of EC cells extends to oxygen sensing.

MicroRNA expression in ileal carcinoid tumors: downregulation of microRNA-133a with tumor progression

MicroRNAs (miRNAs) are involved in cell proliferation, differentiation, and apoptosis and can function as tumor suppressor genes or oncogenes. The role of miRNAs in neuroendocrine tumors such as ileal carcinoids is largely unknown. We examined the differential expression of 95 miRNAs by RT–PCR using the QuantiMir System in eight matching primary and metastatic carcinoid tumors from the ileum. All miRNAs chosen for the QuantiMir System array were based on their potential functions related to cancer biology, cell development, and apoptosis. The expression of miRNAs for the samples was normalized to miRNA-197, and the matching primary and metastatic tumors were compared. There was downregulation of miRNA-133a, -145, -146, -222, and -10b in all samples between the primary and matching metastatic tumors and upregulation of miRNA-183, -488, and -19a+b in six of eight metastatic carcinoids compared to the primary tumors. miRNA-133a was further analyzed by TaqMan real-time RT–PCR and northern hybridization using six additional matching primary and metastatic samples, which supported the PCR array findings. There were significant differences in miRNA-133a expression with downregulation in the metastasis compared to the primary in the eight original cases (P<0.009) and in the six additional cases used for validation (P<0.014). Laser capture microdissection and real-time RT–PCR analysis using normal ileum found miRNA-133a expression in normal enterochromaffin cells. In situ hybridization in normal ileum showed that some of the mucosal endocrine cells expressed miRNA-133a. Both primary and metastatic ileal carcinoid tumors expressed miRNA-133a by in situ hybridization. These results provide information about novel marker miRNAs that may be used as biomarkers and/or therapeutic targets in intestinal carcinoid tumors.

IL1β‐ and LPS‐induced serotonin secretion is increased in EC cells derived from Crohn’s disease

Gut mucosal enterochromaffin (EC) cells are regarded as key regulators of intestinal motility and fluid secretion via secretion of serotonin (5HT), are increased in numbers in mucosal inflammation and located in close proximity to immune cells. We examined whether interleukin (IL)1beta and Escherichia coli lipopolysaccharide (LPS) induced EC cell 5HT release through Toll-like/IL-1 (TIL) receptor activation, nuclear factor kappa B (NFkappaB) and mitogen-activated protein kinase (MAPK) phosphorylation and evaluated whether somatostatin could inhibit this phenomenon. Pure (>98%) human intestinal EC cells were isolated by fluorescent activated cell sorting from preparations of normal (n = 5) and Crohn's colitis (n = 6) mucosa. 5HT release was measured (ELISA), and NFkappaB and ERK phosphorylation quantitated (ELISA) in response to IL1beta and LPS. 5HT secretion was increased by both E. coli LPS (EC(50) = 5 ng mL(-1)) and IL1beta (EC(50) = 0.05 pmol L(-1)) >2-fold (P < 0.05) in Crohn's EC cells compared with normal EC cells. Secretion was reversible by the TLR4 antagonist, E. coli K12 LPS (IC(50) = 12 ng mL(-1)) and the IL1beta receptor antagonist (ILRA; IC(50) = 3.4 ng mL(-1)). IL1beta caused significant (P < 0.05) NFkappaB and MAPK phosphorylation (40-55%). The somatostatin analogue, lanreotide inhibited IL1beta-stimulated secretion in Crohn's (IC(50) = 0.61 nmol L(-1)) and normal EC cells (IC(50) = 1.8 nmol L(-1)). Interleukins (IL1beta) and bacterial products (E. coli LPS) stimulated 5HT secretion from Crohn's EC cells via TIL receptor activation (TLR4 and IL1beta). Immune-mediated alterations in EC cell secretion of 5HT may represent a component of the pathogenesis of abnormal bowel function in Crohn's disease. Inhibition of EC cell-mediated 5HT secretion may be an alternative therapeutic strategy in the amelioration of inflammatory bowel disease symptomatology.

Novel markers for enterochromaffin cells and gastrointestinal neuroendocrine carcinomas

The gene expression profile of metastasizing serotonin-producing neuroendocrine carcinomas, which arise from enterochromaffin cells in the jejunum and ileum, is still largely unknown. The aim of this study was to identify genes and proteins, which are preferentially expressed by neuroendocrine carcinoma and enterochromaffin cells and therefore potential novel biomarkers and/or therapeutic targets. Six carcinoma specimens and six normal ileal mucosas were profiled by Affymetrix microarrays. Advanced bioinformatics identified differentially and specifically expressed genes, which were validated by quantitative real-time-PCR on tumor cells extracted by laser capture microdissection and normal enterochromaffin cells extracted by immunolaser capture microdissection. We identified six novel marker genes for neuroendocrine carcinoma cells: paraneoplastic antigen Ma2 (PNMA2), testican-1 precursor (SPOCK1), serpin A10 (SERPINA10), glutamate receptor ionotropic AMPA 2 (GRIA2), G protein-coupled receptor 112 (GPR112) and olfactory receptor family 51 subfamily E member 1 (OR51E1). GRIA2 is specifically expressed by neuroendocrine carcinoma cells whereas the others are also expressed by normal enterochromaffin cells. GPR112 and OR51E1 encode proteins associated with the plasma membrane and may therefore become targets for antibody-based diagnosis and therapy. Hierarchical clustering shows high similarity between primary lesions and liver metastases. However, chemokine C-X-C motif ligand 14 (CXCL14) and NK2 transcription factor related locus 3 Drosophila (NKX2-3) are expressed to a lower level in liver metastases than in primary tumors and normal enterochromaffin cells, which implies a role in neuroendocrine carcinoma differentiation. In conclusion, this study provides a list of genes, which possess relatively specific expression to enterochromaffin and neuroendocrine carcinoma cells and genes with differential expression between primary tumors and metastases. We verified six novel marker genes that may be developed as biomarkers and/or therapeutic targets.

44 Abnormal Immune-Mediated (IL1β/LPS) Regulation of Small Intestinal EC Cell Serotonin Release in Inflammatory Bowel Disease

Background: Inflammatory bowel disease (IBD) is a common but troublesome disease with limited treatment options. The mechanisms behind IBD-associated mucosal damage and secretory andmotility abnormalities are complex and ill-defined. The gut mucosal enterochromaffin (EC) cell is regarded as a key regulator of intestinal motility and fluid secretion via its secretion of serotonin. It has recently been demonstrated that immune cells are located in close proximity to EC cells in the intestinal mucosa. Furthermore, EC cell numbers are increased in gut inflammation, and as gut hypermobility and hypersecretion are typical of IBD symptoms, we postulated that an activated immune system may lead to EC cell dysregulation and hypersecretion of serotonin. We further hypothesized that both immune derived cytokines (IL1β) and lipopolysaccharides (LPS) from bacteria may induce EC cell activation. LPS signaling is known to involve NFκβ activation through binding to TOLL receptor 4 (TLR4). Methods and results: Pure (>98%) human intestinal EC cells were isolated by FACS sorting from preparations of normal (n=5) and IBD (n=6) mucosa. Using real time PCR, a 5-fold higher TLR4 (p 2-fold (p<0.05) in IBD EC cell cultures compared to normal EC cells. This was not due to cell damage (<1% alteration in Trypan blue or MTT uptake) and was reversible by the TLR4 antagonist, E. coli K12 (IC50=12ng/ml) and the IL1β receptor antagonist (ILRA; IC50=3.4ng/ml) respectively. Addition of IL1β to IBD EC cells also resulted in significant (p<0.05) NFκβ phosphorylation (30-40%). The somatostatin analogue, lanreotide (somatostatin receptor profile: 2,3,5), inhibited IL1β-stimulated secretion in both IBD EC (IC50= 0.61nM) and normal EC cells (IC50=1.8nM). In conclusion, serotonin release from EC cells isolated from IBDmucosa is increased more potently by both interleukins (IL1β) and bacterial products (E. coli LPS) compared to normal EC cells. This occurs through a mechanism that involves NFκβ phosphorylation and can be inhibited by somatostatin analogues and a TLR4 antagonist. Identifying and delineating an abnormal EC cell function is important in elucidating the pathogenesis behind hypersecretion in IBD. In addition, investigating the role of the EC cell will provide the potential for an alternative therapeutic target to reduce symptoms in IBD.

Small bowel carcinoid (enterochromaffin cell) neoplasia exhibits transforming growth factor–β1‐mediated regulatory abnormalities including up‐regulation of C‐Myc and MTA1

Although it is known that small intestinal carcinoids are derived from enterochromaffin (EC) cells, these cells remain poorly characterized and little is known of the growth regulatory mechanisms of these neuroendocrine cells. Down-regulation or loss of the transforming growth factor-beta1 (TGFbeta1) cytostatic program and activation of TGFbeta-mediated transcriptional networks is associated with uncontrolled growth and metastasis in other neural tumors, glioblastomas. Whether this phenomenon is common to small intestinal carcinoid tumors was investigated. The effects of TGFbeta1 on cultured normal EC cells (isolated by FACS sorting) and the neoplastic EC cell line, KRJ-I, was assessed using the MTT assay. The TGFbetaRII transcript and protein were identified in tumor cells and the effects of TGFbeta1 on SMAD2 phosphorylation and nuclear translocation quantified. The time-dependent response of SMAD4, SMAD7, c-Myc, and P21(WAF1/CIP1) protein expression and c-Myc and p21(WAF1/CIP1) transcript was measured in response to TGFbeta1 and the transcript expression of candidate downstream targets, MTA1 and E-cadherin, were assessed. TGFbeta1 inhibited normal EC cell proliferation (IC(50) = 17 pM) but stimulated neoplastic EC cell proliferation (EC(50) = 22 pM). In tumor cells, significantly decreased transcript (P < .01) of TGFbetaRII was identified, but no receptor mutations were identified and protein expression was evident. TGFbeta1 (1 ng/mL) resulted in SMAD2 phosphorylation and <7% nuclear expression compared with 93% in normal EC cells. In neoplastic cells, TGFbeta1 (1 ng/mL) caused a decrease in SMAD4 (>16%, P < .05), whereas SMAD7 and c-Myc transcript and protein were respectively increased >21% (P < .05) and approximately 40% (P < .002). TGFbeta1 (1 ng/mL) also decreased p21(WAF1/CIP1) transcript by 60% (P < .001) and protein that was undetectable at 24 hours. Expression of the downstream targets of the c-Myc pathway, MTA1, was increased (20%) and E-cadherin decreased (30%). The neoplastic EC cell is characterized by loss of TGFbeta-1-mediated growth inhibition and, similar to glioblastomas, utilizes the TGFbeta system to induce gene responses associated with growth promotion (c-Myc and the ERK pathway), invasion (E-cadherin), and metastasis (MTA1).

Further delineation of the continuous human neoplastic enterochromaffin cell line, KRJ-I, and the inhibitory effects of lanreotide and rapamycin

Small intestinal carcinoids (SICs) are the most prevalent gastrointestinal carcinoid and characterized by local invasion metastasis and protean symptomatology. The proliferative and secretory regulation of the cell of origin, the enterochromaffin (EC) cell has not been characterized. The absence of either a pure preparation of normal EC cells or human EC carcinoid cell lines has hindered the development of therapeutic agents. We therefore further characterized the neoplastic SIC cell line, KRJ-I by assessing its secretory (serotonin (5-HT)) and proliferative responses and defining its log growth phase transcriptome. Electron microscopy demonstrated oval, lobulated nuclei and substance P, and 5-HT-positive cytoplasmic vesicles. RT-PCR detected transcripts for chromogranin A (CHGA), VMAT1 (SLC18A1), tryptophan hydroxylase (TPH1), substance P (TAC1), guanylin (GUCA2A), and SERT (SLC6A4). By immunohistochemistry, all cells were positive for CHGA, SERT, VMAT1, and TPH1. Transcriptome analysis (Affymetrix U133 Plus chips) identified somatostatin SSTR2/3, adrenergic alpha1C and beta1, dopamine D2, nicotinic-type cholinergic A5, A6, B1, muscarinic acetylcholine M4, and 5-HT-2A receptors. The presence of transcripts for SSTR1, SSTR2, and SSTR3 receptors was confirmed by RT-PCR and sequencing. Isoproterenol (ISO) resulted in a dose-dependent increase in intracellular cAMP (EC50=340 nM) and 5-HT (EC50=81 nM) which was completely inhibited by the cAMP antagonist 2',5'-dideoxyadenosine (10 microM). Preincubation with a SSTR agonist, lanreotide, inhibited Ip-stimulated 5-HT secretion (IC50=420 nM). Both lanreotide (10 nM) and rapamycin (50 nM) inhibited proliferation (20+/-12 and 35+/-5% respectively) in serum-free medium whereas gefitinib (1 nM-10 microM) inhibited proliferation at micromolar concentrations. KRJ-I is a neoplastic EC cell line that can be used as an in vitro model of SICs as it will allow elucidation and clarification of the secretory and proliferative mechanism(s) of neoplastic EC cells and the molecular signatures that characterize each of these responses.

Association of DNA methylation and epigenetic inactivation of RASSF1A and beta-catenin with metastasis in small bowel carcinoid tumors

We analyzed promoter methylation of RASSF1A, CTNNB1, CDH1, LAMB3, LAMC2, RUNX3, NORE1A, and CAV1 using methylation-specific PCR in 33 cases of small bowel carcinoid with both matched primary and metastatic tumors. The methylation status of RASSF1A and CTNNB1 were also determined in six primary appendiceal carcinoid tumors. Two neuroendocrine cell lines, NCI-H727 and HTB-119, were analyzed for promoter methylation. Immunohistochemical analyses for RASSF1A and beta-catenin were performed in 28 matched primary and metastatic tumors. Western blot analysis for RASSF1A and beta-catenin was also performed. Normal enterochromaffin cells were unmethylated in all eight genes examined. RASSF1A and CTNNB1 were unmethylated in appendiceal carcinoids. Methylation of RASSF1A and CTNNB1 promoters was more frequent in metastatic compared to primary tumors (p = 0.013 and 0.004, respectively). The NCI-H727 and HTB-119 cells lines were methylated in the RASSF1A promoter region, and after treatment with 5-aza-2'-deoxycytidine (5-AZA), RASSF1A mRNA was expressed in both cell lines. Western blot results for RASSF1A and beta-catenin supported the methylation-specific PCR findings. The other six genes did not show significant differences. These results suggest that increased methylation of RASSF1A and CTNNB1 may play important roles in progression and metastasis of small bowel carcinoid tumors.

Transmembrane protein tyrosine phosphatase IA-2 (ICA512) is expressed in human midgut carcinoids but is not detectable in normal enterochromaffin cells.

A potential upregulation of receptor type protein tyrosine phosphatase IA-2 (ICA512) expression was detected by differential display and investigated in midgut carcinoid tumours. Normal intestine tissue and tumour tissue from 13 midgut carcinoid patients were studied by in situ hybridisation using an IA-2 ribonucleotide probe and confocal microscopy using specific IA-2 antibodies. Previously, it had been shown that IA-2 is located in the secretory granules of virtually all neuroendocrine cells. However, we found that IA-2 was not detectable in resting normal enterochromaffin (EC) cells of the small intestine, while high expression of IA-2 mRNA and protein was confirmed in both primary and metastatic carcinoid tissue. This difference in expression was not observed with chromogranin A or serotonin, two secretory granule hormones known to be expressed in EC cells, indicating that IA-2 was seemingly not necessary for the basal production and packaging of these hormones. When comparing patients receiving biotherapy before operation with untreated patients, we found expression of IA-2 to be lower in tumours from patients that had been treated with a combination of alpha-interferon and the somatostatin analogue, octreotide. There was no correlation between IA-2 expression and proliferation rates as measured by immunohistochemistry with antibodies against the Ki 67 antigen. Furthermore, we show that IA-2 is co-localised with serotonin in carcinoid tumours as well as in the pancreatic tumour cell line, BON1, which is interesting as serotonin secretion rate is presumably higher in tumour cells than in resting EC cells. Taken together, these findings may indicate a role for IA-2 in the later stages of the regulated secretory process.

Ultrastructural characterization of enterochromaffin-type cells in human gastric carcinoma.

Ultrastructural characterization has been performed on eosinophilic granular cells occurring in gastric carcinomas. Of these, the present paper dealt with enterochromaffin (EC)-type cells. EC-type cells were identified by the presence of eosinophilic granules, immunoreactivity for serotonin (5 HT) and argentaffinity. Conventional electron microscopy revealed that they bore membrane bound, electron-dense granules of varying size (100-800 nm in shorter diameter), which were larger than those of normal EC cells. Their shapes are round, rod-shaped and concave. The granules are immunolabeled for 5HT by both pre- and post-embedding methods. The preembedding method also disclosed diffuse cytosolic immunoreactivity. Both differentiated-type and undifferentiated-type gastric carcinomas showed essentially the same results. General trends of occurrence of Paneth-like and EC-type cells in gastric carcinomas were also discussed. The present study confirmed that EC-type cells in gastric carcinoma retained basic characteristics of EC cells despite of morphological deviation from the normal cells. It is speculated that they are derived from neoplastic precursor cells with a special differentiation.

Investigations on the structure of the enterochromaffin substance.

Summarizing, a catechol structure for enterochromaffin seems more indicated than a hydroquinone structure. Any aminophenol struture, primary or secondary, seems contraindicated. The resorcinol hypothesis seems untenable. Serotonin is chemically and sometimes histochemically demonstrable in carcinoid tumors, but its presence in normal enterochromaffin cells in demonstrable amounts seems contraindicated. The azo positive phenol in the enterochromaffin cells appears to be a distinct substance.