ECL-cell carcinoids and carcinoma in patients homozygous for an inactivating mutation in the gastric H(+) K(+) ATPase alpha subunit.

A family with a missense variant of the ATP4A gene encoding the alpha subunit of the gastric proton pump (H(+) K(+) ATPase) has recently been described. Homozygous siblings were hypergastrinemic (median gastrin 486 pM) and had gastric tumours diagnosed at a median age of 33 years. In the current histopathological study, we further characterized the tumours found in the gastric corpus. The tumours had the histological appearance of carcinoids (NET G1 or G2) and were immunoreactive for the general neuroendocrine markers chromogranin A (CgA) and synaptophysin as well as the ECL-cell markers vesicular monoamine transporter 2 (VMAT2) and histidine decarbozylase (HDC). One of the tumours consisted of a NET G2 component, but also had a component with glandular growth, which morphologically was classified as an intestinal type adenocarcinoma. Many glands of the adenocarcinoma contained a large proportion of cells positive for neuroendocrine markers, especially the small vesicle marker synaptophysin and the cytoplasmic enzyme HDC. In conclusion, patients homozygous for an inactivating ATP4A mutation develop gastric ECL-cell carcinoids in their 3rd or 4th decade. The adenocarcinoma may be classified as neuroendocrine with ECL-cell differentiation.

Large-scale evaluation of SLC18A2 in prostate cancer reveals diagnostic and prognostic biomarker potential at three molecular levels.

Limitations of current diagnostic and prognostic tools for prostate cancer (PC) have led to over-diagnosis and over-treatment. Here, we investigate the biomarker potential of the SLC18A2 (VMAT2) gene for PC at three molecular levels. Thus, SLC18A2 promoter methylation was analyzed in 767 malignant and 78 benign radical prostatectomy (RP) samples using methylation-specific qPCR and Illumina 450K methylation microarray data. SLC18A2 transcript levels were assessed in 412 malignant and 45 benign RP samples using RNAseq data. SLC18A2 protein was evaluated by immunohistochemistry in 502 malignant and 305 benign RP samples. Cancer-specificity of molecular changes was tested using Mann-Whitney U tests and/or receiver operating characteristic (ROC) analyses. Log rank, uni- and multivariate Cox regression tests were used for survival analyses. We found that SLC18A2 promoter hypermethylation was highly cancer-specific (area under the curve (AUC): 0.923-0.976) and associated with biochemical recurrence (BCR) after RP in univariate analyses. SLC18A2 transcript levels were reduced in PC and had independent prognostic value for BCR after RP (multivariate HR 0.13, P < 0.05). Likewise, SLC18A2 protein was down-regulated in PC (AUC 0.898) and had independent prognostic value for BCR (multivariate HR 0.51, P < 0.05). Reduced SLC18A2 protein expression was also associated with poor overall survival in univariate analysis (HR 0.29, P < 0.05). Our results highlight SLC18A2 as a new promising methylation marker candidate for PC diagnosis. Furthermore, SLC18A2 expression (RNA and protein) showed promising prognostic potential beyond routine clinicopathological variables. Thus, novel SLC18A2-based molecular tests could have useful future applications for PC detection and identification of high-risk patients.

Histidine decarboxylase and urinary methylimidazoleacetic acid in gastric neuroendocrine cells and tumours.

AIM: To study histidine decarboxylase (HDC) expression in normal and neoplastic gastric neuroendocrine cells in relationship to the main histamine metabolite. METHODS: Control tissues from fundus (n = 3) and corpus (n = 3) mucosa of six patients undergoing operations for gastric adenocarcinoma, biopsy and/or gastric surgical specimens from 64 patients with primary gastric neuroendocrine tumours (GNETs), as well as metastases from 22 of these patients, were investigated using conventional immunohistochemistry and double immunofluorescence with commercial antibodies vs vesicular monoamine transporter 2 (VMAT-2), HDC and ghrelin. The urinary excretion of the main histamine metabolite methylimidazoleacetic acid (U-MeImAA) was determined using high-performance liquid chromatography in 27 of the 64 patients. RESULTS: In the gastric mucosa of the control tissues, co-localization studies identified neuroendocrine cells that showed immunoreactivity only to VMAT-2 and others with reactivity only to HDC. A third cell population co-expressed both antigens. There was no co-expression of HDC and ghrelin. Similar results were obtained in the foci of neuroendocrine cell hyperplasia associated with chronic atrophic gastritis type A and also in the tumours. The relative incidence of the three aforementioned markers varied in the tumours that were examined using conventional immunohistochemistry. All of these GNETs revealed both VMAT-2 and HDC immunoreactivity, and their metastases showed an immunohistochemical pattern and frequency similar to that of their primary tumours. In four patients, increased U-MeImAA excretion was detected, but only two of the patients exhibited related endocrine symptoms. CONCLUSION: Human enterochromaffin-like cells appear to partially co-express VMAT-2 and HDC. Co-expression of VMAT-2 and HDC might be required for increased histamine production in patients with GNETs.

The gastric mucosa 25 years after proximal gastric vagotomy.

OBJECTIVE: Vagotomy causes inhibition of basal and post-prandial acid secretion in humans, but the knowledge about the trophic effect of the vagal nerves is limited. Vagotomy is known to induce hypergastrinemia and we aimed to study the long-term effects of proximal gastric vagotomy (PGV) on the oxyntic mucosa and the enterochromaffin-like (ECL) cell density in particular. MATERIAL AND METHODS: Eleven patients operated with PGV because of duodenal ulcer and age- and sex-matched controls were examined 26 to 29 years postoperatively by gastroscopy with biopsies from the antrum and oxyntic mucosa. Neuroendocrine cell volume densities were calculated after immunohistochemical labeling of gastrin, the general neuroendocrine cell marker chromogranin A (CgA) and the ECL cell marker vesicular monoamine transporter 2 (VMAT2). Gastritis was graded and Helicobacter pylori (H. pylori) status was determined by polymerase chain reaction of gastric biopsies. Fasting serum gastrin and CgA were measured. RESULTS: Serum gastrin was higher in the PGV group compared to controls (median 21.0 [interquartile range (IQR) = 22.0] pmol/L vs 13.0 [IQR = 4.0] pmol/L, p = 0.04). However, there was neither a significant difference in serum CgA or in CgA (neuroendocrine) nor VMAT2 (ECL cell) immunoreactive cell volume density in the oxyntic mucosa. There was significantly more inflammation and atrophy in H. pylori-positive patients, but PGV did not influence the grade of gastritis. CONCLUSION: Despite higher serum gastrin concentrations, patients operated with PGV did not have higher ECL cell mass or serum CgA. Vagotomy may prevent the development of ECL cell hyperplasia caused by a moderate hypergastrinemia.

Obestatin/ghrelin cells in normal mucosa and endocrine tumours of the stomach.

OBJECTIVE: Obestatin and ghrelin are derived from the same gene and co-expressed in the same endocrine cells. Vesicular monoamine transporter-2 (VMAT-2), a marker for enterochromaffin-like (ECL) cells, is considered to be expressed in ghrelin cells. The aim was to establish if the two peptides and the transporter are co-expressed, both in normal gastric mucosa and in gastric endocrine tumours. DESIGN: An immunohistochemical study was performed on gastric biopsy material and on surgical specimens from 63 patients with gastric endocrine tumours and from individuals with normal gastric mucosa. Cells displaying obestatin immunoreactivity were examined regarding co-localization with ghrelin and VMAT-2. Both single- and double-immunostaining techniques were applied. Obestatin concentration in blood was measured in a subgroup of these patients. The results were correlated to various clinico-pathological parameters. RESULTS: In the normal mucosa, obestatin/ghrelin-immunoreactive cells rarely co-expressed VMAT-2. In most tumour tissue specimens, only a fraction of neoplastic cells displayed immunoreactivity to obestatin, and these cells always co-expressed ghrelin. Neoplastic obestatin-/ghrelin-IR cells invariably expressed VMAT-2, except for two ghrelinomas. The obestatin concentrations in blood were consistently low and did not correlate to clinico-pathological data. CONCLUSIONS: Obestatin and ghrelin immunoreactivity always occurred in the same endocrine cells in the gastric mucosa but these cells only occasionally co-expressed VMAT-2, opposite to the findings in tumours. These results indicate that endocrine cells expressing obestatin and ghrelin mainly differ from VMAT-2 expressing cells (ECL-cells) and can develop into pure ghrelinomas. Plasma concentrations of obestatin did not correlate to cellular expression.

Genetic and epigenetic SLC18A2 silencing in prostate cancer is an independent adverse predictor of biochemical recurrence after radical prostatectomy.

PURPOSE: This study investigates SLC18A2 (vesicular monoamine transporter 2) expression in prostate adenocarcinoma and examines its potential as a predictive marker for prostate cancer patient outcome after radical prostatectomy. EXPERIMENTAL DESIGN: Expression and single nucleotide polymorphism microarray analyses identified SLC18A2 as both down-regulated and subject to common loss-of-heterozygosity in prostate cancer. Down-regulated SLC18A2 expression was validated on tissue microarrays containing benign and malignant prostate specimens from an independent patient group (n=738). Furthermore, SLC18A2 immunoreactivity in radical prostatectomy tumor specimens (n=506) was correlated to clinicopathologic characteristics and recurrence-free survival. The possibility of SLC18A2 silencing by aberrant DNA methylation in prostate cancer cells was investigated by bisulfite sequencing. RESULTS: Tissue microarray analysis revealed markedly lower cytoplasmic SLC18A2 staining in cancer compared with nonmalignant prostate tissue samples, confirming RNA expression profiling results. Furthermore, multivariate analysis identified cytoplasmic SLC18A2 immunoreactivity as a novel predictor of biochemical recurrence following prostatectomy (hazard ratio, 0.485; 95% confidence interval, 0.333-0.709; P<0.001) independent of prostate-specific antigen, Gleason score, tumor stage, and surgical margin status. SLC18A2 showed loss-of-heterozygosity in 23% of the tumors and was densely hypermethylated in 15 of 17 (88%) prostate cancer samples plus 6 of 6 prostate cancer cell lines. In contrast, SLC18A2 was unmethylated in 4 of 4 adjacent nonmalignant prostate and 3 of 5 benign prostatic hyperplasia tissue samples, whereas 2 of 5 benign prostatic hyperplasia samples had monoallelic hypermethylation. Methylation and histone deacetylase inhibitory agents rescued SLC18A2 expression in three prostate cancer cell lines. CONCLUSIONS: SLC18A2 silencing by DNA hypermethylation and/or allelic loss is a frequent event in prostate cancer and a novel independent predictor of biochemical recurrence after prostatectomy.

Methylphenidate-induced increases in vesicular dopamine sequestration and dopamine release in the striatum: the role of muscarinic and dopamine D2 receptors.

Methylphenidate (MPD) administration alters the subcellular distribution of vesicular monoamine transporter-2 (VMAT-2)-containing vesicles in rat striatum. This report reveals previously undescribed pharmacological features of MPD by elucidating its receptor-mediated effects on VMAT-2-containing vesicles that cofractionate with synaptosomal membranes after osmotic lysis (referred to herein as membrane-associated vesicles) and on striatal dopamine (DA) release. MPD administration increased DA transport into, and decreased the VMAT-2 immunoreactivity of, the membrane-associated vesicle subcellular fraction. These effects were mimicked by the D2 receptor agonist quinpirole and blocked by the D2 receptor antagonist eticlopride. Both MPD and quinpirole increased vesicular DA content. However, MPD increased, whereas quinpirole decreased, K(+)-stimulated DA release from striatal suspensions. Like MPD, the muscarinic receptor agonist, oxotremorine, increased K(+)-stimulated DA release. Both eticlopride and the muscarinic receptor antagonist scopolamine blocked MPD-induced increases in K(+)-stimulated DA release, whereas the N-methyl-d-aspartate receptor antagonist (-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) was without effect. This suggests that D2 receptors mediate both the MPD-induced redistribution of vesicles away from synaptosomal membranes and the MPD-induced up-regulation of vesicles remaining at the membrane. This results in a redistribution of DA within the striatum from the cytoplasm into vesicles, leading to increased DA release. However, D2 receptor activation alone is not sufficient to mediate the MPD-induced increases in striatal DA release because muscarinic receptor activation is also required. These novel findings provide insight into the mechanism of action of MPD, regulation of DA sequestration/release, and treatment of disorders affecting DA disposition, including attention-deficit hyperactivity disorder, substance abuse, and Parkinson's disease.

Mitochondrial complex I inhibitor rotenone inhibits and redistributes vesicular monoamine transporter 2 via nitration in human dopaminergic SH-SY5Y cells.

Parkinson's disease is a progressive neurodegenerative disorder characterized by selective degeneration of nigrostriatal dopaminergic neurons. Long-term systemic mitochondrial complex I inhibition by rotenone induces selective degeneration of dopaminergic neurons in rats. We have reported dopamine redistribution from vesicles to the cytosol to play a crucial role in selective dopaminergic cell apoptosis. In the present study, we investigated how rotenone causes dopamine redistribution to the cytosol using an in vitro model of human dopaminergic SH-SY5Y cells. Rotenone stimulated nitration of the tyrosine residues of intracellular proteins. The inhibition of nitric-oxide synthase or reactive oxygen species decreased the amount of nitrotyrosine and attenuated rotenone-induced apoptosis. When we examined the intracellular localization of dopamine immunocytochemically using anti-dopamine/vesicular monoamine transporter 2 (VMAT2) antibodies and quantitatively using high-performance liquid chromatography, inhibiting nitration was found to suppress rotenone-induced dopamine redistribution from vesicles to the cytosol. We demonstrated rotenone to nitrate tyrosine residues of VMAT2 using an immunocytochemical method with anti-nitrotyrosine antibodies and biochemically with immunoprecipitation experiments. Rotenone inhibited the VMAT2 activity responsible for the uptake of dopamine into vesicles, and this inhibition was reversed by inhibiting nitration. Moreover, rotenone induced the accumulation of aggregate-like formations in the stained image of VMAT2, which was reversed by inhibiting nitration. Our findings demonstrate that nitration of the tyrosine residues of VMAT2 by rotenone leads to both functional inhibition and accumulation of aggregate-like formations of VMAT2 and consequently to the redistribution of dopamine to the cytosol and apoptosis of dopaminergic SH-SY5Y cells.

Mitochondrial complex I inhibitor rotenone-elicited dopamine redistribution from vesicles to cytosol in human dopaminergic SH-SY5Y cells.

Parkinson's disease is a chronic neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra. Rotenone, a pesticide, produces selective degeneration of dopaminergic neurons and motor dysfunction in rats. To determine the mechanisms underlying rotenone-induced neuronal death, we investigated whether intracellular dopamine plays a role in rotenone (0.1-0.4 microM)-induced apoptosis, using an in vitro model of human dopaminergic SH-SY5Y cells. The 40% decrease of dopamine content by inhibition of dopamine synthesis suppressed rotenone-induced apoptosis. On the other hand, the 30% increase of dopamine content by inhibition of dopamine metabolism enhanced rotenone-induced apoptosis. Depletion of intracellular dopamine using reserpine (0.1-10 microM) also prevented rotenone-induced apoptosis, and this effect was counteracted by dopamine (10-100 microM) replenishment. Inhibition of dopamine reverse transport increased cytosolic dopamine and enhanced rotenone-induced apoptosis. We examined the intracellular localization of dopamine in rotenone-treated cells immunocytochemically and quantitatively. Rotenone induced dopamine redistribution from vesicles to the cytosol. In this process, rotenone stimulated reactive oxygen species and protein carbonylation and decreased an antioxidant, glutathione. Addition of an antioxidant, N-acetylcysteine (3 mM), prevented dopamine being expelled from vesicles and inhibited rotenone-induced apoptosis. Our findings demonstrate that rotenone-generated reactive oxygen species are involved in dopamine redistribution to the cytosol, which in turn may play a role in rotenone-induced apoptosis of dopaminergic cells.