Manabu Wada

Isolation and Characterization of a GDP/GTP Exchange Protein Specific for the Rab3 Subfamily Small G Proteins

The Rab small G protein family, consisting of nearly 30 members, is implicated in intracellular vesicle trafficking. They cycle between the GDP-bound inactive and GTP-bound active forms, and the former is converted to the latter by the action of a GDP/GTP exchange protein (GEP). No GEP specific for each Rab family member or Rab subfamily has been isolated. Here we purified a GEP from rat brain with lipid-modified Rab3A as a substrate. The purified protein was specifically active on Rab3A, Rab3C, and Rab3D of the Rab3 subfamily. Of these subfamily members, Rab3A and Rab3C are implicated in Ca2+-dependent exocytosis, particularly in neurotransmitter release. This GEP (Rab3 GEP) was active on the lipid-modified form, but not on the lipid-unmodified form. Rab3 GEP showed a minimum molecular mass of about 200 kDa on SDS-polyacrylamide gel electrophoresis. We cloned its cDNA from a rat brain cDNA library and determined its primary structure. The isolated cDNA encoded a protein with a Mr of 177,982 and 1,602 amino acids, which showed no homology to any known protein. The recombinant protein exhibited GEP activity toward Rab3A, Rab3C, and Rab3D. Northern blot and Western blot analyses indicated that Rab3 GEP was expressed in all the rat tissues examined with the highest expression in brain.

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.

Restoration of RUNX3 enhances transforming growth factor-β-dependent p21 expression in a biliary tract cancer cell line

RUNX3 is a candidate tumor suppressor gene localized in 1p36, a region commonly inactivated by deletion and methylation in various human tumors. To elucidate the role of RUNX3 in transforming growth factor (TGF)‐β signaling in biliary tract cancer, we transfected Mz‐ChA‐2 cells, which do not express RUNX3 but have intact TGF‐β type II receptor and SMAD4 genes, with the RUNX3 expression plasmid pcDNA3.1/RUNX3 or with the vector pcDNA3.1 as a control. Four Mz‐ChA‐2/RUNX3 clones and one control clone were obtained. Although TGF‐β1 only slightly inhibited growth of the control cells, growth inhibition and TGF‐β‐dependent G1 arrest were significantly enhanced in the RUNX3‐transfected clones. None of the clones, however, exhibited apoptosis. The slightly increased TGF‐β1‐induced p21 expression in the control clone was strongly enhanced in the RUNX3‐transfected clones, and was accompanied by augmented decreases in the expression of cyclins D1 and E. When RUNX3 small interfering RNA was added, TGF‐β‐dependent induction of p21 was reduced in the RUNX3‐transfected clones. Xenografts of the clones in nude mice demonstrated that tumorigenicity was significantly decreased in the RUNX3‐transfected clones in inverse proportion to the expression levels of RUNX3. Based on these results, RUNX3 is involved in TGF‐β‐induced expression of p21 and the resulting induction of TGF‐β‐dependent G1 arrest. (Cancer Sci 2007; 98: 838–843)

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.

Endoscopic ultrasonograph evaluation of vascular structures in the gastric cardia predicts esophageal variceal recurrence following endoscopic treatment

Background and Aim:  In patients with portal hypertension, early recurrence of esophageal varices often occurs following endoscopic variceal ligation therapy or ligation and injection‐sclerotherapy combined treatment. To assess the recurrence risk following endoscopic treatment, this study investigated the association between recurrence‐free time and severity of esophagogastric vascular structures before treatment as determined by endoscopic ultrasonography.

Improvement of Collateral Vessels in the Vicinity of Gastric Cardia After Endoscopic Variceal Ligation Therapy for Esophageal Varices

BACKGROUND & AIMS Endoscopic variceal ligation (EVL) therapy has been performed widely to treat or prevent variceal bleeding. We sought to examine the influence of EVL for esophageal varices on collateral vessels in the vicinity of gastric cardia. METHODS In 42 patients with esophagogastric varices, conventional endoscopy and endoscopic ultrasonography with a 20-MHz probe (CUP-EUS) were performed before and at every 3 months after EVL for esophageal varices. By using conventional endoscopy, cardial variceal sizes were divided into 3 grades: F0, F1, and F2. The sizes of submucosal, perforating, and paracardial vessels at the cardia also were classified into 3 grades according to CUP-EUS findings. RESULTS Conventional endoscopy showed cardial varices in 33 (79%) patients before and 23 (55%) patients at 3 months after the treatment (P < 0.05). CUP EUS showed that 29 (69%) patients had severe grade cardial submucosal vessels before EVL, but only 13 (31%) patients did after the treatment (P < 0.01). Nineteen (45%) patients had severe grade cardial perforating vessels before EVL, but only 4 (10%) patients did after the treatment (P < 0.001). Furthermore, patients with severe grade residual submucosal or perforating vessels at the cardia had shorter recurrence-free times of esophageal varices (P < 0.01, 0.05, respectively). CONCLUSIONS Collateral vessels in the vicinity of gastric cardia were improved significantly after EVL, indicating that esophageal varices can be treated by EVL even though they connect with cardial varices. Furthermore, eradication of such collateral vessels by EVL may lead to longer recurrence-free status of esophageal varices.