Mitsutaka Sawada

Candidate markers for stem and early progenitor cells, Musashi‐1 and Hes1, are expressed in crypt base columnar cells of mouse small intestine

Musashi‐1, a neural RNA‐binding protein, is important for maintaining neural stem cells. Both Musashi‐1 and Hes1, a transcriptional factor regulated by Musashi‐1, are expressed in the small intestine. Here we show that Musashi‐1 is present in a few epithelial cells just above the Paneth cells in the small intestinal crypt, the putative position of stem cells, whereas Hes1 is expressed in lower crypt cells just above the Paneth cells, including Musashi‐1‐positive cells. Musashi‐1 and Hes1 were not expressed in Paneth cells. Notably, Musashi‐1 and Hes1 were coexpressed in the crypt base columnar cells located between the Paneth cells. These findings suggest that not only the cells just above Paneth cells but also the crypt base columnar cells between the Paneth cells have stem cell characteristics.

STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells

Signal transduction and activator of transcription 3(STAT3) signaling is constitutively activated in various tumors, and is involved in cell survival and proliferation during oncogenesis. There are few reports, however, on the role of STAT3 signaling in gastric cancer. The aim of the present study was to clarify the role of STAT3 signaling in apoptosis and cellular proliferation in gastric cancer. Here we reported that STAT3 was constitutively activated in various human gastric cancer cells and its inhibition by ectopic dominant-negative STAT3 or Janus kinase inhibitor, tyrphostin AG490, induced apoptosis. Furthermore, STAT3 inhibition markedly decreased survivin expression, and forced expression of survivin rescued AGS cells from apoptosis induced by STAT3 inhibition. Although some reports demonstrated that the PI3K/Akt pathway regulates survivin expression, inhibition of the PI3K/Akt pathway did not affect survivin expression in AGS and MKN1 cells. Finally, activated form of STAT3, Tyr-705 phospho-stat3, was found in the nucleus of cancer cells in 11 of 40 (27.5%) human gastric cancer specimens. These findings suggest that constitutively activated STAT3 signaling supports gastric cancer cell survival in association with survivin expression.

Frequent loss of RUNX3 gene expression in human bile duct and pancreatic cancer cell lines

RUNX3, a Runt domain transcription factor involved in TGF-β signaling, is a candidate tumor-suppressor gene localized in 1p36, a region commonly deleted in a wide variety of human tumors, including those of the stomach, bile duct, and pancreas. Recently, frequent inactivation of RUNX3 has been demonstrated in human gastric carcinomas. In this study, to examine the involvement of RUNX3 abnormalities in tumorigenesis of bile duct as well as pancreatic cancers, we investigated not only the expression but also methylation status of RUNX3 in 10 human bile duct and 12 pancreatic cancer cell lines. Seven (70%) of the bile duct and nine (75%) of the pancreatic cancer cell lines exhibited no expression of RUNX3 by both Northern blot analysis and the reverse transcriptase polymerase chain reaction. All of the 16 cell lines that did not express RUNX3 also showed methylation of the promoter CpG island of the gene, whereas the six cell lines that showed RUNX3 expression were not methylated or only partially methylated in the RUNX3 promoter region. Moreover, treatment with the methylation inhibitor 5′-aza-2′-deoxycitidine activated RUNX3 mRNA expression in all of 16 cancer cell lines that originally lacked RUNX3 expression. Finally, hemizygous deletion of RUNX3, as detected by fluorescence in situ hybridization, was found in 15 of the 16 cancer cell lines that lacked RUNX3 expression. These data suggest that the inactivation of RUNX3 plays an important role in bile duct and pancreatic carcinogenesis, and that methylation is a common mechanism by which the gene is inactivated.

Comparison of the signal transduction pathways activated by gastrin in enterochromaffin-like and parietal cells

BACKGROUND & AIMS Gastrin stimulates acid secretion from parietal cells and histamine release from enterochromaffin-like (ECL) cells through identical gastrin receptors. However, gastrin has been shown to have a trophic effect only on ECL cells. The aim of this study was to compare gastrin-induced signal transduction pathways in the ECL and parietal cells of Mastomys natalensis, an African rodent. METHODS Both ECL and parietal cells were isolated from the gastric mucosa of M. natalensis, and intracellular signal transduction events in response to gastrin were investigated. RESULTS Gastrin elicited histamine release from ECL cells and acid secretion from parietal cells in association with enhanced inositol phospholipid turnover. Although gastrin increased [3H]thymidine incorporation into ECL cells, it had no effect on parietal cells. Moreover, tyrosine phosphorylation and activation of mitogen-activated protein (MAP) kinase as well as c-fos and c-jun gene expression were augmented only in ECL cells. In addition, gastrin increased the formation of guanosine triphosphate-Ras with a simultaneous decrease in guanosine diphosphate-Ras levels in ECL but not in parietal cells. CONCLUSIONS Although gastrin receptors are present in both ECL and parietal cells, they activate the Ras-MAP kinase pathway only in ECL cells.

Expression of Reg Iα Protein in Human Gastric Cancers

Background/Aims: Although regeneratinggene(Reg) Iα protein has a trophic effect on gastric epithelial cells, it is unclear whether Reg Iα protein and its receptor are involved in gastric carcinogenesis. Therefore, we investigated the Reg Iα protein expression in human gastric cancers and assessed its relationship to clinicopathological factors. Methods: Sixty-one gastric cancer specimens were examined, using immunohistochemistry, for Reg Iα protein, p53, and proliferating cell nuclear antigen. The expression of both Reg Iα and Reg receptor mRNA was examined in seven human gastric cancer cell lines (MKN1, MKN28, MKN45, MKN74, KATOIII, GCIY, and AGS) by reverse transcription-polymerase chain reaction and Northern blot analysis. Results: Twenty-three (37.7%) of the 61 gastric cancer tissues samples were positive for Reg Iα protein. The Reg Iα expression was significantly related to the presence of lymphatic invasion but not to tumor size, tumor stage, Lauren’s classification, presence of venous invasion, lymph node metastases, or p53 overexpression. Gastric cancers positive for Reg Iα protein showed a significantly higher proliferating cell nuclear antigen labeling index than negative ones. The expression of both Reg Iα and Reg receptor mRNA was detected in all seven gastric cancer cell lines. Conclusion: Reg Iα protein may play a role in the development of gastric cancers.

Induction of follicular gastritis following postthymectomy autoimmune gastritis in Helicobacter pylori-infected BALB/c mice

ABSTRACT Helicobacter pylori is the major causative agent of chronic antral gastritis and is thought to be involved in the pathogenesis of mucosa-associated lymphoid tissue lymphoma (MALToma) developing in the human stomach. The aim of this study was to clarify whether corporal autoimmune gastritis (AIG), which is known to decrease acidity due to destruction of parietal cells, predisposes mice toH. pylori infection, thereby leading to MALToma-like pathology. BALB/c mice in which AIG had been induced by thymectomy 3 days after birth (AIG mice) were used. The AIG mice were orally administered mouse-adapted H. pylori at the age of 6 weeks and were examined histologically and serologically after 2 to 12 months. The results were compared with those obtained from uninfected AIG mice and infected normal mice. Germinal centers were induced in the corpus in 57% of the H. pylori-infected AIG mice, which elicited anti-H. pylori antibody responses in association with upregulation of interleukin-4 (IL-4) mRNA. In these mice, parietal cells remained in the corpus mucosa. These findings were in contrast to those with the uninfected AIG mice: fundic gland atrophy due to disappearance of parietal cells associated with upregulation of gamma interferon, but not IL-4, mRNA and no germinal center formation in the corpus. These observations suggest that AIG alters the infectivity ofH. pylori, leading to MALToma-like follicular gastritis, at an early stage after H. pylori infection.

Involvement of Tumor Necrosis Factor Alpha in Intestinal Epithelial Cell Proliferation Following Paneth Cell Destruction

Background : An intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induces selective killing of Paneth cells which have a large amount of zinc in their cytoplasmic granules. A transient wave of intestinal epithelial cell proliferation occurs at 12 h after the injection. Paneth cells have tumor necrosis factor (TNF)- α protein in their cytoplasmic granules, and TNF- α has a proliferative effect on intestinal epithelial cells in vitro. The aim of this study is to clarify the in vivo role of TNF- α in intestinal epithelial cell proliferation using a dithizone-treated rat model. Methods : Male Wistar rats received a dithizone (100 mg/kg of body weight) injection with or without TNF- α inhibitor, pentoxifylline (100 mg/kg), neutralizing anti-TNF- α antibody (2 mg/kg), or nuclear transcription factor s B (NF- s B) inhibitors; pyrrolidine dithiocarbamate (100 mg/kg) or N -acetyl-L-cystein (100 mg/kg). The activation of NF- s B was examined by the electrophoretic mobility shift assay, and cellular proliferation by BrdU labeling. Results : Without any inhibitors, dithizone treatment evoked NF- s B activation in the ileal mucosa with its peak level at 2 h after the injection. TNF- α inhibition reduced the NF- s B activation, and blocked a transient wave of epithelial cell proliferation 12 h after the injection. NF- s B inhibitors also reduced the NF- s B activation and epithelial cell proliferation. Conclusions : TNF- α released from degenerated Paneth cells was, in part, responsible for the intestinal cell proliferation through the activation of NF- s B, suggesting its proliferative effect on intestinal epithelial cells.

Identification of xanthine dehydrogenase/xanthine oxidase as a rat Paneth cell zinc-binding protein

Paneth cells are zinc-containing cells localized in small intestinal crypts, but their function has not been fully elucidated. Previously, we showed that an intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induced selective killing of Paneth cells, and purified a zinc-binding protein in Paneth cells. In the present study, we further characterized one of these proteins, named zinc-binding protein of Paneth cells (ZBPP)-1. Partial amino acid sequences of ZBPP-1 showed identity with rat xanthine dehydrogenase (XD)/xanthine oxidase (XO). Anti-rat XD antibody (Ab) recognized ZBPP-1, and conversely anti ZBPP-1 Ab recognized 85 kDa fragment of rat XD in Western blotting. Messenger RNA and protein levels of XD were consistent with our previous data on the fluctuation of Paneth cell population after dithizone injection. Thus, ZBPP-1 is an 85 kDa fragment of XD/XO in Paneth cells. XD/XO in Paneth cells may play important roles in intestinal function.

Cyclooxygenase-2 expression and angiogenesis in gastric hyperplastic polyp - Association with polyp size

Background: Cyclooxygenase (COX)-2 is the rate-limiting enzyme in prostaglandin synthesis, and plays an important role in tumor enlargement. COX-2 is expressed in human gastric and colorectal tumors, and the expression increases in a tumor size-dependent manner. In the present study, we attempted to examine the COX-2 expression pattern in gastric hyperplastic polyp, a non-tumorous lesion. Patients and Methods: Fifty-eight gastric hyperplastic polyps, obtained by endoscopic polypectomy, were immunostained with anti-COX-2 and antivascular endothelial growth factor (VEGF) antibodies. Microvessel density (MVD) was determined by von Willebrand factor immunostaining. Results: In larger gastric hyperplastic polyps, COX-2 was expressed mainly on the luminal side of the polyp stroma, while it was absent in smaller polyps. A significant correlation between COX-2 immunoreactivity and polyp size was observed (p < 0.01). High VEGF expression and MVD were observed mainly in the same stromal region of the polyps where COX-2 was expressed. Both VEGF expression and MVD were also correlated with polyp size significantly (ps < 0.01). Conclusions: COX-2 expression increased in a size-dependent manner in non-tumorous hyperplastic polyps, suggesting that COX-2 expression is not necessarily linked to epithelial cell transformation. Moreover, COX-2 may participate in polyp enlargement through angiogenesis by promoting VEGF production.

Refractory enterovesical and duodenocolic fistulas in Crohn's disease successfully managed with tacrolimus.

the present patient, the area showing a hepatoid appearance was immunohistochemically negative for PIVKA-II. To our knowledge, only five cases of PIVKA-II-producing gastric cancer, including the present case, have been reported in the English-language literature.2–5 The result of immunohistochemical staining for PIVKA-II was poor in two patients, partially positive in two patients, and positive in one patient. Four (80%) out of the five patients were positive for hepatic metastasis, causing death within 6 months. PIVKA-II is a putative tumor marker of HCC; however, when tumors are found in the stomach and liver, and the serum PIVKA-II level is abnormally high, the possibility of PIVKA-II-producing gastric cancer with liver metastasis should be considered.