Gastric Epithelial Cell Turnover Research Articles

MTOR signaling regulates gastric epithelial progenitor homeostasis and gastric tumorigenesis via MEK1-ERKs and BMP-Smad1 pathways.

mTOR, the sensor of nutrients and growth factors, has important roles in tissue homeostasis and tumorigenesis. However, how mTOR controls gastric epithelial cell turnover and gastric cancer development, a leading malignancy, remains poorly understood. Here, we provide genetic evidence that mTOR activation promotes proliferation and inhibits differentiation of Lgr5+ gastric epithelial progenitors (GEPs) in gastric homeostasis and tumorigenesis. mTOR signaling increases MEK1 and Smad1 expression and enhances activation of MEK1-ERKs and BMP-Smad1 pathways, respectively, in GEPs and gastric tumors. Mek1 deletion or inhibition rescues hyperproliferation, whereas Bmpr1a ablation or inhibition rescues differentiation defects of Tsc1-/- GEPs. Tsc1 deficiency in Lgr5+ GEPs accelerates gastric tumor initiation and development, which require MEK1-ERKs for hyperplasia and BMP-Smad1 for differentiation suppression. These findings reveal how mTOR signaling controls Lgr5+ GEP homeostasis and cancerization and suggest that ERKs and Smad1 signaling can be safely targeted to substitute mTOR inhibitors in gastric cancer therapy.

Hexane Extracts of Calophyllum brasiliense Inhibit the Development of Gastric Preneoplasia in Helicobacter felis Infected INS-Gas Mice.

Objectives: Indigenous Latin American populations have used extracts from Calophyllum brasiliense, a native hardwood, to treat gastrointestinal symptoms for generations. The hexane extract of Calophyllum brasiliense stem bark (HECb) protects against ethanol-mediated gastric ulceration in Swiss–Webster mice. We investigated whether HECb inhibits the development of gastric epithelial pathology following Helicobacter felis infection of INS-Gas mice.Materials and Methods: Groups of five male, 6-week-old INS-Gas mice were colonized with H. felis by gavage. From 2 weeks after colonization their drinking water was supplemented with 2% Tween20 (vehicle), low dose HECb (33 mg/L, lHECb) or high dose HECb (133 mg/L, hHECb). Equivalent uninfected groups were studied. Animals were culled 6 weeks after H. felis colonization. Preneoplastic pathology was quantified using established histological criteria. Gastric epithelial cell turnover was quantified by immunohistochemistry for Ki67 and active-caspase 3. Cytokines were quantified using an electrochemiluminescence assay.Results: Vehicle-treated H. felis infected mice exhibited higher gastric atrophy scores than similarly treated uninfected mice (mean atrophy score 5.6 ± 0.87 SEM vs. 2.2 ± 0.58, p < 0.01). The same pattern was observed following lHECb. Following hHECb treatment, H. felis status did not significantly alter atrophy scores. Gastric epithelial apoptosis was not altered by H. felis or HECb administration. Amongst vehicle-treated mice, gastric epithelial cell proliferation was increased 2.8-fold in infected compared to uninfected animals (p < 0.01). Administration of either lHECb or hHECb reduced proliferation in infected mice to levels similar to uninfected mice. A Th17 polarized response to H. felis infection was observed in all infected groups. hHECb attenuated IFN-γ, IL-6, and TNF production following H. felis infection [70% (p < 0.01), 67% (p < 0.01), and 41% (p < 0.05) reduction vs. vehicle, respectively].Conclusion: HECb modulates gastric epithelial pathology following H. felis infection of INS-Gas mice. Further studies are indicated to confirm the mechanisms underlying these observations.

PTH-165 Hexane extracts of calophyllum brasiliense inhibit the development of gastric preneoplasia in helicobacter felis infected ins-gas mice

Introduction Calophyllum brasiliense is a tropical hardwood tree native to Latin America. Indigenous populations have used extracts from the stem bark of this tree to treat gastrointestinal symptoms for generations. In previous studies we demonstrated that a hexane extract of Calophyllum brasiliense stem bark (HECb) protects against an acidified ethanol model of gastric ulceration in Swiss-Webster mice. We hypothesised that HECb would also inhibit the gastric epithelial pathology, including gastric preneoplasia that is induced by Helicobacter infection. To investigate this we have used the established hypergastrinaemic INS-Gas, H. felis infection model. Method Groups of 6 male, 6 week old INS-Gas mice were colonised with H. felis by oral gavage. Between 2 weeks after colonisation and the end of the procedure their drinking water was supplemented with 2% Tween 20, HECb 83.3 mg/L (lHECb) or HECb 333.3mg/L (hHECb). Equivalent uninfected control groups were also studied. Animals were culled 6 weeks after H. felis colonisation. Gastric mucosal samples were taken for histology and protein extraction. Preneoplastic pathology was quantified using established histological criteria. Gastric epithelial cell turnover was quantified by immunohistochemistry for Ki67 and active-caspase 3. Cytokines were quantified using electrochemiluminescence assays. Results H. felis infected mice treated with Tween 20 exhibited increased gastric atrophy scores than uninfected mice treated with Tween 20 (mean atrophy score 5.6+/-0.87 SEM vs 2.2+/-0.58 , p H. felis or HECb administration. Compared to uninfected mice, gastric epithelial cell proliferation was increased 2.8 fold in infected, Tween 20 treated mice (p h 17 polarised response to H. felis infection and decreased abundance of IFN-γ, IL-6 and TNF in the infected mice administered hHECb (70% (p vs Tween 20 respectively). Conclusion HECb modulates gastric epithelial pathology following H. felis infection in INS-Gas mice. The extract influences both epithelial cell turnover and cytokine production. Further studies are indicated to dissect the mechanisms underlying these observations, and to identify the biologically active constituents of HECb. Disclosure of interest None Declared.

Helicobacter pyloriInfection Induces Oxidative Stress and Programmed Cell Death in Human Gastric Epithelial Cells

Helicobacter pylori infection is associated with altered gastric epithelial cell turnover. To evaluate the role of oxidative stress in cell death, gastric epithelial cells were exposed to various strains of H. pylori, inflammatory cytokines, and hydrogen peroxide in the absence or presence of antioxidant agents. Increased intracellular reactive oxygen species (ROS) were detected using a redox-sensitive fluorescent dye, a cytochrome c reduction assay, and measurements of glutathione. Apoptosis was evaluated by detecting DNA fragmentation and caspase activation. Infection with H. pylori or exposure of epithelial cells to hydrogen peroxide resulted in apoptosis and a dose-dependent increase in ROS generation that was enhanced by pretreatment with inflammatory cytokines. Basal levels of ROS were greater in epithelial cells isolated from gastric mucosal biopsy specimens from H. pylori-infected subjects than in cells from uninfected individuals. H. pylori strains bearing the cag pathogenicity island (PAI) induced higher levels of intracellular oxygen metabolites than isogenic cag PAI-deficient mutants. H. pylori infection and hydrogen peroxide exposure resulted in similar patterns of caspase 3 and 8 activation. Antioxidants inhibited both ROS generation and DNA fragmentation by H. pylori. These results indicate that bacterial factors and the host inflammatory response confer oxidative stress to the gastric epithelium during H. pylori infection that may lead to apoptosis.

Altered expression of Skp2, c-Myc and p27 proteins but not mRNA after H. pylori eradication in chronic gastritis

Helicobacter pylori infection is associated with increased gastric epithelial cell turnover and non-cardia gastric cancer. Cell cycle progression is dependent on the proteasomal degradation of p27, a cyclin-dependent kinase inhibitor and gastric tumor suppressor, following ubiquitination mediated by Skp2. c-Myc is a transcriptional repressor of p27 and also a target of Skp2. In vitro, H. pylori decreases p27 protein post-translationally. We aimed to determine how p27 is regulated by H. pylori in vivo. The effect of eradicating H. pylori on gastric epithelial p27, Skp2, and c-Myc proteins and mRNA was investigated in 22 patients with chronic gastritis, by immunohistochemistry and laser capture microdissection. The percentage of gastric antral epithelial cells expressing p27 protein was significantly higher after eradication of H. pylori (mean±s.e.m. 37±2.4% pre-eradication vs 55±2.8% post-eradication; P<0.001), while Skp2 and c-Myc protein-expressing cells were lower (Skp2: 35±3.8 vs 23±2.6%, P=0.009; c-Myc: 47±3.6 vs 30±3.8%, P<0.001). mRNA expressions of p27, Skp2, and c-Myc (normalized for 18SrRNA) were not changed by H. pylori eradication. H. pylori increases c-Myc and decreases gastric epithelial p27 protein expression in association with increased expression of Skp2, the regulator of p27’s ubiquitin ligase complex. H. pylori may influence cell cycle progression and carcinogenesis through post-translational effects on specific gene expression.

Gastric epithelial cell turnover and mucosal protection in Japanese children with Helicobacter pylori infection

In adults, epithelial cell proliferation and apoptosis of the gastric mucosa are induced by Helicobacter pylori infection and are associated with gastric atrophy or gastric carcinoma. In children, there are few studies about such epithelial changes. To elucidate the role of H. pylori infection in gastric mucosal inflammation, we immunohistochemically examined gastric mucosa of Japanese children. Biopsy specimens obtained from the gastric antrum and corpus of H. pylori-infected (n = 13) and noninfected children (n = 15) were studied for immunolocalization of Ki-67, single-strand DNA, manganese superoxide dismutase (Mn-SOD), and CD68, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling. In 10 patients with successful eradication, pre- and posttreatment results were compared. In both gastric antrum and corpus, neutrophil and mononuclear cell infiltration, epithelial cell proliferation, and apoptosis significantly increased in H. pylori-infected patients, predominantly in the antrum. In the antrum of H. pylori-infected patients, there was positive correlation between the degrees of neutrophil infiltration and cell proliferation (P < 0.05) or apoptosis (P < 0.05). H. pylori eradication improved mucosal inflammation, cell proliferation (P < 0.001), and apoptosis (P < 0.01) in the antrum. Mn-SOD immunoreactivity and CD68-positive macrophages in the antrum, which significantly increased in H. pylori-infected patients, decreased after the eradication. H. pylori infection induced gastric mucosal inflammation and epithelial cell turnover in children. Moreover, gastric mucosal defense mechanism against H. pylori infection was activated. H. pylori eradication in childhood might prevent the accumulation of gastric epithelial cell damage.

Effects of Helicobacter pylori infection on cell cycle progression and the expression of cell cycle regulatory proteins.

Helicobacter pylori lives in the stomach lumen adhering and specifically interacting with gastric epithelial cells. H. pylori infection can cause a broad range of diseases. Although most infected individuals only develop a chronic inflammation of the stomach, some patients progress to chronic gastritis, duodenal ulceration, or, rarely, cancer. H. pylori is able to send and to receive signals from the gastric epithelium, allowing host and bacteria to become linked in a dynamic equilibrium. Several studies have demonstrated that H. pylori infection induces morphological changes of gastric epithelial cells other than cell proliferation, increase of mitosis and mutations. It has also been demonstrated that H. pylori may predispose to cancer by altering gastric epithelial cell turnover acting specifically on transcription factors. Although H. pylori is able to induce several host responses, it specifically perturbs the delicate balance of those factors that usually help to maintain cell homeostasis. The study of mechanisms of interaction between the bacterium and gastric cells will surely help to prevent the increase and diffusion of malignancies all over the world.

Helicobacter pylori Releases a Factor(s) Inhibiting Cell Cycle Progression of Human Gastric Cell Lines by Affecting Cyclin E/cdk2 Kinase Activity and Rb Protein Phosphorylation through Enhanced p27 KIP1 Protein Expression

Helicobacter pylori, the main cause of chronic gastritis, plays a central role in the etiology of peptic ulcer disease and gastric cancer. In vitro studies have shown that H. pylori increases gastric epithelial cell turnover, thus increasing the risk for the development of neoplastic clones. The mechanisms by which H. pylori promotes perturbation of cell proliferation are not yet elucidated. To investigate whether products released by H. pylori in culture media interfere with cell cycle progression of human gastric epithelial cells, four cell lines (MKN 28, MKN 7, MKN 74, and AGS) were incubated in the presence of H. pylori broth culture filtrate. Cell cycle analysis showed that a H. pylori-released factor(s) significantly inhibited the G1- to S-phase progression of MKN 28 and MKN 7 cell lines, with a reversible, nonlethal mechanism, independent of the expression of VacA, CagA, and/or urease. The cell cycle inhibition occurred concomitantly with an increase in p27 KIP1 protein levels, a reduction in Rb protein phosphorylation on serine residues 807–811, and a significant decrease in cyclin E-associated cdk2 activity. In contrast, the cell cycle progression of MKN 74 and AGS cell lines was not affected by the H. pylori-released factor(s). In normal human fibroblasts, G1-phase cell accumulation was concomitant with the reduction in Rb protein phosphorylation; that, however, appeared to be dependent on p21 WAF1/CIP1 rather than on p27 KIP1 protein. A preliminary characterization showed that the molecular mass of the partially purified cell cycle inhibitory factor(s) was approximately 40 kDa. These results suggest that H. pylori releases a soluble factor(s) that may affect cell cycle progression of gastric epithelial cells through elevated levels of cdk inhibitor p27 KIP1. This factor(s) might act in vivo on noncolonized distant cells, the most proliferating cells of human gastric mucosa.

Enhanced Expression of Transcription Factor E2F in Helicobacter pylori‐infected Gastric Mucosa

Helicobacter pylori is implicated in gastric carcinogenesis through increased gastric epithelial cell turnover. In fact, high proportions of proliferating and apoptotic epithelial cells are found in H. pylori-infected gastric mucosa. E2F, a transcription factor, induces coordinated transactivation of a set of genes involved in cell cycle progression. The aim of this study was to investigate the expression of E2F in H. pylori-infected gastric mucosa and examine the correlation between such expression and gastric epithelial cell proliferation and apoptosis. Twenty-five patients with H. pylori-associated gastritis (HAG) and 13 control subjects negative for H. pylori were examined. E2F expression was studied in situ by Southwestern histochemistry, a method used to localize transcription factors. Labeled double-stranded oligo-DNA with specific consensus sequence for E2F binding sites was reacted with frozen sections from antral biopsy specimens obtained at endoscopy. Gastric epithelial cell proliferation was assessed by immunostaining of proliferating cell nuclear antigen (PCNA), while apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The percentages of epithelial cells with nuclear staining for PCNA and E2F were expressed as a positivity index (PI). The percentage of TUNEL-positive epithelial cells was defined as apoptotic index. E2F was expressed in the nuclei of gastric epithelial cells within gastric pits. E2F PI in H. pylori-infected gastric mucosa was significantly higher than that in noninfected. Expression of E2F correlated well with PCNA-positive epithelial cells. We also demonstrated colocalization of PCNA with E2F expression in the same epithelial cells. Apoptotic index was also high in H. pylori-infected mucosa, and correlated with E2F PI. Our results demonstrated a significant increase in the expression of E2F in H. pylori-infected mucosa, which correlated with both the percentages of PCNA- and TUNEL-positive cells. Our results suggest that enhanced E2F expression in gastric mucosa may be involved in H. pylori-related gastric carcinogenesis through accelerated cell turnover.

Helicobacter pylori alters gastric epithelial cell cycle events and gastrin secretion in Mongolian gerbils

Background & Aims: Human colonization with Helicobacter pylori increases the risk for distal gastric adenocarcinoma, possibly by altering gastric epithelial cell cycle events and/or gastrin secretion. This study aimed to determine whether H. pylori virulence–related characteristics affect apoptosis, proliferation, and gastrin levels in a rodent model of gastric adenocarcinoma. Methods: Mongolian gerbils were challenged with H. pylori wild-type or isogenic cagA − and vacA − mutants, and apoptotic and proliferating cells were identified by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling and proliferating cell nuclear antigen immunohistochemistry, respectively. Serum gastrin levels were determined by radioimmunoassay. Results: Gastric epithelial cell turnover was no different after infection with the wild-type, cagA − , or vacA − strains. H. pylori infection significantly increased antral apoptosis 2-4 weeks after challenge, before apoptotic indices decreased to baseline. In contrast, antral proliferation rates were significantly higher 16-20 weeks after inoculation, but then decreased by 40 weeks. Antral proliferation was significantly related to serum gastrin levels, whereas antral apoptosis was inversely related to acute inflammation and lymphoid follicles. Conclusions: In H. pylori–infected gerbils, enhanced antral apoptosis is an early and transient cell cycle event. Epithelial cell proliferation peaks later and is significantly related to increased gastrin levels, suggesting that epithelial cell growth in H. pylori–colonized mucosa may be mediated by gastrin-dependent mechanisms. GASTROENTEROLOGY 2000;118:48-59

Non-Steroidal Anti-Inflammatory Drugs and Gastric DNA Synthesis

In vitro incubation of endoscopic gastric antral biopsy specimens with tritiated thymidine, with confirmatory autoradiography, was used to assess gastric epithelial cell turnover in man and to investigate the effects of chronic administration of non-steroidal anti-inflammatory drugs (NSAIDs). In the absence of NSAID administration, macroscopic erosions and ulceration in the antrum were associated with enhanced gastric epithelial cell proliferation. In patients taking NSAIDs erosions occurred without a compensatory increase in epithelial cell DNA synthesis. The failure to respond to injury by enhancing epithelial cell proliferation is likely to be an important factor in NSAID-induced gastric damage and supports a role for prostaglandins in enhancing mucosal repair by increasing epithelial cell generation.

Gastric epithelial cell turnover, mucus production, and healing of gastric ulcers with carbenoxolone.

Nineteen healthy subjects were studied and 17 patients with gastric ulcer before and after ulcer healing with carbenoxolone. Gastric deoxytibonucleic acid (DNA) loss was measured as an index of epithelial cell turnover, and N-acetylneuraminic acid (NANA) content of gastric juice as an index of mucus secretion. In normal subjects there was a negative correlation (p less that 0-025) between gastric DNA loss and NANA secretion; the lower the cell turnover the higher the NANA production. In gastric ulcer patients DNA loss or turnover was significantly (p less than 0-01) higher than normal, and fell significantly (p less than 0-01) after four weeks' treatment with carbenoxolone when 16 of the 17 ulcers had healed. At the same time NANA output increased significantly (p less than 0-01). It is suggested that patients with gastric ulcer lose cells at a high rate, a state of affairs which is returned towards normal by carbenoxolone, thus allowing the epithelial cells to mature within the mucosa and produce more mucus.