12 research outputs found
Optimization of cDNA microarrays procedures using criteria that do not rely on external standards-5
<p><b>Copyright information:</b></p><p>Taken from "Optimization of cDNA microarrays procedures using criteria that do not rely on external standards"</p><p>http://www.biomedcentral.com/1471-2164/8/377</p><p>BMC Genomics 2007;8():377-377.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2147032.</p><p></p
Optimization of cDNA microarrays procedures using criteria that do not rely on external standards-4
<p><b>Copyright information:</b></p><p>Taken from "Optimization of cDNA microarrays procedures using criteria that do not rely on external standards"</p><p>http://www.biomedcentral.com/1471-2164/8/377</p><p>BMC Genomics 2007;8():377-377.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2147032.</p><p></p> lines NRK52E and AR42J) compared to self-self hybridization (rat cell line AR42J). The samples were hybridized to rat 15 k cDNA duplicates under six different blocking conditions including no blocker, 1000 ng poly(dA), and 25 to 1000 ng LNA dT blocker. Dye-swap and self self were performed for all blocking conditions (total of 24 hybridizations). Green-labelled samples are placed at the tail and red labelled samples at the head of the arrows
Optimization of cDNA microarrays procedures using criteria that do not rely on external standards-1
<p><b>Copyright information:</b></p><p>Taken from "Optimization of cDNA microarrays procedures using criteria that do not rely on external standards"</p><p>http://www.biomedcentral.com/1471-2164/8/377</p><p>BMC Genomics 2007;8():377-377.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2147032.</p><p></p
The cytoprotective protein clusterin is overexpressed in hypergastrinemic rodent models of oxyntic preneoplasia and promotes gastric cancer cell survival
<div><p>The cytoprotective protein clusterin is often dysregulated during tumorigenesis, and in the stomach, upregulation of clusterin marks emergence of the oxyntic atrophy (loss of acid-producing parietal cells)-associated spasmolytic polypeptide-expressing metaplasia (SPEM). The hormone gastrin is important for normal function and maturation of the gastric oxyntic mucosa and hypergastrinemia might be involved in gastric carcinogenesis. Gastrin induces expression of clusterin in adenocarcinoma cells. In the present study, we examined the expression patterns and gastrin-mediated regulation of clusterin in gastric tissue from: humans; rats treated with proton pump (H+/K+-ATPase) inhibitors and/or a gastrin receptor (CCK2R) antagonist; H+/K+-ATPase β-subunit knockout (H/K-β KO) mice; and Mongolian gerbils infected with <i>Helicobacter pylori</i> and given a CCK2R antagonist. Biological function of secretory clusterin was studied in human gastric cancer cells. Clusterin was highly expressed in neuroendocrine cells in normal oxyntic mucosa of humans and rodents. In response to hypergastrinemia, expression of clusterin increased significantly and its localization shifted to basal groups of proliferative cells in the mucous neck cell-chief cell lineage in all animal models. That shift was partially inhibited by antagonizing the CCK2R in rats and gerbils. The oxyntic mucosa of H/K-β KO mice contained areas with clusterin-positive mucous cells resembling SPEM. In gastric adenocarcinomas, clusterin mRNA expression was higher in diffuse tumors containing signet ring cells compared with diffuse tumors without signet ring cells, and clusterin seemed to be secreted by tumor cells. In gastric cancer cell lines, gastrin increased secretion of clusterin, and both gastrin and secretory clusterin promoted survival after starvation- and chemotherapy-induced stress. Overall, our results indicate that clusterin is overexpressed in hypergastrinemic rodent models of oxyntic preneoplasia and stimulates gastric cancer cell survival.</p></div
Clusterin and TFF2 expression in oxyntic mucosa of Mongolian gerbils increase after <i>H</i>. <i>pylori</i> infection and normalize when antagonizing the CCK2R.
<p>(A, B) Number per oxyntic area (0.44 mm<sup>2</sup>) of (A) CLU-positive/VMAT2(neuroendocrine (NE))-positive cells and CLU-positive/VMAT2(NE)-negative cells and (B) dual CLU-positive/Ki67-positive cells from uninfected control (n = 4), <i>H</i>. <i>pylori</i>-infected (<i>H</i>. <i>pylori</i>) (n = 7), and CCK2R-antagonized <i>H</i>. <i>pylori</i>-infected gerbils (<i>H</i>. <i>pylori</i>+CCK2Ra) (n = 4). (C, D) Oxyntic expression of CLU (brown) and dual CLU-positive (green)/VMAT2-positive (red) ECL cells in (C) uninfected control (n = 4) and (D) CCK2R-antagonized <i>H</i>. <i>pylori</i>-infected gerbils (n = 4). (E) ISH and IHC showing expression of clusterin in oxyntic mucosa of <i>H</i>. <i>pylori</i>-infected gerbils (n = 7). (F) Double immunofluorescence staining showing co-expression of CLU (green) and TFF2 (red, upper figure) and CLU (green) and Ki67 (red, lower figure) in oxyntic mucosa of <i>H</i>. <i>pylori</i>-infected gerbils. (G) IHC showing TFF2 expression (brown) in oxyntic mucosa of uninfected controls and <i>H</i>. <i>pylori</i>-infected gerbils untreated and treated with a CCK2R antagonist. Data presented as means ± SEM. *ANOVA with Tukey-adjusted <i>p</i> value < 0.05. Nuclei were counterstained with hematoxylin (blue) or DAPI (blue). The basal zone (~100 μm from the gland bottom) is highlighted with a dotted line. Scale bars = (C, D left column, E left column, G) 200 μm; (E right column, F upper figure) 100 μm; (D right column, F lower figure) 50 μm.</p
Clusterin and mucous neck cell markers are co-localized in oxyntic mucosa of H/K-β KO mice.
<p>(A, B) ISH and IHC showing overexpression of clusterin (brown) in oxyntic mucosa of H/K-β KO mice aged 8 months (n = 4) (middle column) and 14 months (n = 4) (right column) compared with wild-type control mice (n = 4) (left column). (C) Immunofluorescence staining showing increased GSII (green) expression in oxyntic mucosa of H/K-β KO mice aged 8 months and 14 months compared with wild-type control mice, particularly apparent in cells located in the basal half of glands. (D) Double immunofluorescence staining showing CLU (green) expression in TFF2-positive cells (red) in oxyntic mucosa from wild-type control mice and H/K-β KO mice aged 8 and 14 months. (E) Double immunofluorescence staining showing co-expression of GSII (green) and PEPII (red) in oxyntic mucosa from H/K-β KO mice aged 3 months. (F) Number of dual CLU-positive/Ki67-positive cells per area (0.44 mm<sup>2</sup>) of oxyntic mucosa (n = 4 mice per group). Data presented as means ± SEM. The basal zone (~100 μm from the gland bottom) is highlighted with a dotted line. *ANOVA with Tukey-adjusted <i>p</i> value < 0.05. Nuclei were counterstained with hematoxylin (blue) or DAPI (blue). Scale bars = 100 μm.</p
Clusterin expression in oxyntic mucosa increases and localization shifts from neuroendocrine cells to chief cells after sustained hypoacidity and hypergastrinemia.
<p>(A) IHC and ISH showing similar expression patterns for clusterin (brown) protein and mRNA, examined in control (n = 8) and hypergastrinemic PPI-rats (n = 6). (B) Number of CLU-positive/VMAT2(neuroendocrine (NE))-positive cells and CLU-positive/VMAT2(NE)-negative cells per oxyntic gland (n = 4 rats per group). (C) Double immunofluorescence staining of oxyntic mucosa from hypergastrinemic PPI-rats showing scarce CLU (green) expression in ECL cells (VMAT2-positive (red)). (D) Double immunofluorescence staining of oxyntic mucosa from hypergastrinemic PPI-rats showing CLU (green) expression in groups of chief cells (MIST1-positive (purple)) and triple immunofluorescence staining showing CLU (green) expression in proliferating (PCNA-positive (light blue)) chief cells (PGA5 (red)). Ctr = control; PPI = PPI-induced hypergastrinemia; CCK2Ra = CCK2R antagonist; PPI+CCK2Ra = PPI-induced hypergastrinemia+CCK2R antagonist. Data presented as means ± SEM. *, ** and # = ANOVA with Bonferroni-adjusted <i>p</i> value < 0.05. (*comparison of CLU+/NE+ cells per gland in control vs other groups individually; **comparison of CLU+/NE- cells per gland in PPI vs control or CCK2Ra; #comparison of CLU+/NE- cells per gland in PPI+CCK2Ra vs CCK2Ra.) Nuclei were counterstained with hematoxylin (blue) or DAPI (blue). The basal zone (~100 μm from the gland bottom) is highlighted with a dotted line. Scale bars = (A) 100 μm; (C, D) 50 μm.</p
Biochemical and morphological characteristics of control rats and rats with PPI-induced hypergastrinemia, CCK2R antagonist treatment alone and PPI-induced hypergastrinemia in combination with CCK2R antagonist treatment.
<p>Biochemical and morphological characteristics of control rats and rats with PPI-induced hypergastrinemia, CCK2R antagonist treatment alone and PPI-induced hypergastrinemia in combination with CCK2R antagonist treatment.</p
Primary antibodies used in immunofluorescence staining and immunohistochemistry.
<p>Primary antibodies used in immunofluorescence staining and immunohistochemistry.</p
Clusterin expression in neuroendocrine cells.
<p>(A) Double immunofluorescence staining of oxyntic mucosa from control rats showing CLU (green) expression in ECL cells (HDC-positive (red)) and A-like cells (ghrelin-positive (red)). A split image of the green (CLU) and red (HDC) fluorescences separately, and a higher magnification of a few dual positive cells (inset), are shown to clearly illustrate the co-expression of CLU in neuroendocrine cells, representative for all species. (B) Double immunofluorescence staining of oxyntic mucosa from control rats showing no CLU (green) expression in parietal cells (H+/K+-ATPase β (HK)-positive (red)). (C) Double immunofluorescence staining of oxyntic mucosa from wild-type control mice showing CLU (red) expression in ECL cells (HDC-positive (green)). (D) Double immunofluorescence staining of oxyntic mucosa from wild-type control mice showing CLU (red) expression in mucous neck cells (GSII-positive (green)). (E) Double immunofluorescence staining of oxyntic mucosa from control Mongolian gerbils showing CLU (green) expression in ECL cells (VMAT2-positive (red)). (F) ISH and IHC of human oxyntic mucosa showing clusterin (brown and green) expression in scattered single cells partially overlapping with the neuroendocrine marker CgA (red). Inset shows high power view of single CLU-positive cells. Nuclei were counterstained with hematoxylin (blue) or DAPI (blue). The basal zone (~100 μm from the gland bottom) is highlighted with a dotted line. Scale bars = (B, D, F middle column) 100 μm; (A, C, E, F left and right column) 50 μm; (F inset) 20 μm.</p