Kenji Morinaka

Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors.

The involvement of Ral and its downstream molecules in receptor‐mediated endocytosis was examined. Expression of either RalG23V or RalS28N, which are known to be constitutively active and dominantnegative forms, respectively, in A431 cells blocked internalization of epidermal growth factor (EGF). Stable expression of RalG23V or RalS28N in CHO‐IR cells also inhibited internalization of insulin. Internalization of EGF and insulin was not affected by full‐length RalBP1 which is an effector protein of Ral, but was inhibited by its C‐terminal region which binds directly to Ral and POB1. POB1 is a binding protein of RalBP1 and has the Eps15 homology (EH) domain. Deletion mutants of POB1 inhibited internalization of EGF and insulin. However, internalization of transferrin was unaffected by Ral, RalBP1, POB1 and their mutants. Epsin and Eps15 have been reported to be involved in the regulation of endocytosis of the receptors for EGF and transferrin. The EH domain of POB1 bound directly to Epsin and Eps15. Taken together with the observation that EGF and insulin activate Ral, these results suggest that Ral, RalBP1 and POB1 transmit the signal from the receptors to Epsin and Eps15, thereby regulating ligand‐dependent receptor‐mediated endocytosis.

Gene mutations of K-ras in gallbladder mucosae and gallbladder carcinoma with an anomalous junction of the pancreaticobiliary duct

ObjectiveIn this study, we examined the mutational spectrum of K-ras in cases of gallbladder and gallbladder carcinoma with an anomalous junction of the pancreaticobiliary duct (AJPBD).MethodsWe examined 35 gallbladders with AJPBD (20 with hyperplasia, 15 with carcinoma) and 38 gallbladders without AJPBD (four normal gallbladders, four with hyperplasia, six with adenoma, 24 with carcinoma). Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to detect mutations in codon 12 or 13 of K-ras.ResultsIn the cases with AJPBD, the prevalences of K-ras mutation were 15% (3/20) in hyperplasia, 60% (6/10) in stage I carcinoma, and 100% (5/5) in stage II–IV carcinoma. In the cases without AJPBD, the prevalences of K-ras mutation were 0% (0/4) in normal gallbladder, 0% (0/4) in hyperplasia, 17% (1/6) in adenoma, 7% (1/16) in stage I carcinoma, and 38% (3/8) in stage II–IV carcinoma. Prevalences of K-ras mutation in hyperplasia and carcinoma with AJPBD were greater than those without AJPBD (p < 0.05). The point mutation of GGT to GAT in codon 12 was frequently observed in the cases with AJPBD.ConclusionsThese results suggest that the specific K-ras mutation in codon 12 (GGT to GAT) may contribute to the early stage of carcinogenesis in the gallbladder with AJPBD.

Epsin binds to the EH domain of POB1 and regulates receptor-mediated endocytosis.

POB1 has been identified as a RalBP1-binding protein and has the Eps15 homology (EH) domain. The EH domain-containing proteins have been suggested to be involved in clathrin-dependent endocytosis. To clarify the function of POB1, we purified a protein which binds to the EH domain of POB1 from bovine brain cytosol and identified it as Epsin, which is known to bind to the EH domain of Eps15. Epsin has three Asn-Pro-Phe (NPF) motifs in the C-terminal region, which are known to form the core sequence for the binding to the EH domain. The EH domain of POB1 interacted directly with the region containing the NPF motifs of Epsin. Expression of Epsin in CHO-IR cells inhibited internalization of insulin although it affected neither insulin-binding nor autophosphorylation activities of the insulin receptor. Taken together with the observations that Epsin is involved in internalization of the receptors for epidermal growth factor and transferrin, these results suggest that Epsin is a binding partner of POB1 and their binding regulates receptor-mediated endocytosis.

Expression of interleukin-8 correlates with invasiveness in human pancreatic cancer

Background Interleukin S (IL-S), a member of the CXC chemokine family, is a multifunctional cytokine shown to induce angiogenesis, haptotactic migration, and proliferation of certain cancer cells. IL-S is presumed to exert its action after binding to its specific receptors (IL-SRA and IL-SRB). Previously, we have reported that matrix metalloproteinase-9 (MMP-9) was increased by treatment with IL-S in gastric cancer cells. The aims of this study was to examine whether IL-S and IL-S receptors were expressed in human pancreatic cancer and to evaluate the role of IL-S on metastatic potential in pancreatic cancer. Methods The expression of IL-S, IL-SRA and IL-SRB in human pancreatic cancer cells (PANC-I, MIA PaCa-2, Capan-2) were examined by northern blot analysis and western blot analysis. The expressions of IL-S and its receptors were also investigated by immunohistochemistry in human pancreatic cancer tissues using specific antibody. Cytokine induced secretions of IL-S in supernatants of cultured medium from cells were investigated with ELISA (R&D). After treatment with various concentrations of recombinant IL-S (Sigma), the cell proliferations were assayed using Cell Counting KitTM (Dojindo Labs., Japan). IL-S induced production and gelatinolytic activity of MMP-2 and MMP-9 were analyzed by western blot analysis and gelatin zymogram using conditioned mediums from each cell as samples. Results The expression of IL-S, IL-SRA and IL-SRB were detected in all investigated pancreatic cancer cells. Moreover, the secretion of IL-S was increased with cytokine stimuli. In addition, immunoreactive signals of IL-8 and its receptors were observed in cancer tissues.· Exogenous adding of IL-S did not induce proliferation of human pancreatic cancer cell lines. Surprisingly, the production and gelatinolytic activity ofMMP-2 and MMP-9 were increased by the treatment of recombinant IL-S in a dose-dependent manner. Conclusion These results suggest that IL-S regulate MMP activity and may play an important role in the invasiveness of human pancreatic cancer.

Cholecystokinin regulates the invasiveness of human pancreatic cancer cell lines via protein kinase C pathway

We have previously reported that cholecystokinin (CCK) plays an important role in the invasiveness and the production of matrix metalloproteinase-9 (MMP-9) in two human pancreatic cancer cell lines. In this study we investigated the pathway of the invasiveness associated with MMP-9 of those lines regulated by CCK. Two human pancreatic cancer cell lines were treated with CCK-8 alone, CCK-8 and staurosporine, or CCK-8 and indomethacine. The invasiveness and the production of MMP-9 were decreased with staurosporine but not indomethacine. These results suggest that CCK may regulate the invasiveness and the production of MMP-9 via protein kinase C in human pancreatic cancer cell lines.

Telomerase activity for the preoperative diagnosis of pancreatic cancer

Among patients with cancer, those with pancreatic cancer have one of the lowest 5-year survival rates (1). Pancreatic cancer is usually diagnosed by ultrasonography, computed tomography, endoscopic ultrasonography, endoscopic retrograde cholangiopancreatography (ERCP), and cytology (2). However, it is sometimes difficult to distinguish benign from malignant tumors with the use of these diagnostic tools (3,4). K-ras mutations in pancreatic secretions have been reported to be useful for the diagnosis of cancer (5) but were also detected in chronic pancreatitis (6). Therefore, K-ras mutations are not considered to be a specific marker for pancreatic cancer. Telomerase is an enzyme that contains an RNA template complementary to the short DNA sequence repeats (GGTTAG in humans) present at chromosomal ends (i.e., telomeres); the enzyme is believed to be involved in the de novo synthesis at those sites (7). A highly sensitive polymerase chain reaction-based telomerase assay called TRAP (Telomeric Repeat Amplification Protocol) was developed (8,9), and led to the detection of telomerase activity in almost all cancer tissues (10–12). Recently, in surgically resected pancreatic tissue specimens, we detected telomerase activity in 41 (95%) of 43 cancer specimens but none (0 of 11) in benign tumor specimens (13). These findings suggest that telomerase activity in pancreatic duct cells may be useful for cancer diagnosis prior to surgery. Therefore, in this study, telomerase was assayed by use of pancreatic duct cells obtained preoperatively. In almost all cases of suspected pancreatic tumors, ERCP is performed and pancreatic duct cells are collected by use of endoscopic retrograde pancreatic duct brushing (ERPDB). ERPDB has been shown to be a useful method for obtaining cells from the sites of pancreatic duct abnormalities (4). From January 1996 through June 1997, pancreatic duct cells were collected by use of ERPDB from 32 patients with pancreatic duct abnormalities at the Hiroshima University Hospital under informed consent. Institutional guidelines for the use of patients’ materials were followed. The 21 male and 11 female patients ranged in age from 34 to 78 years (mean, 62 years). Part of each specimen was processed for cytologic analysis with the use of Pap staining and was then examined by a pathologist (F. Shimamoto) who made a diagnosis according to guidelines reported by Robins et al. (14). After washing the remaining cells with phosphate-buffered saline, the TRAP assay was performed as described previously (13). In a preliminary study, we examined telomerase activity without making an adjustment for cell number, and weak telomerase activity was detected in three of three chronic pancreatitis cases with massive infiltration of lymphocytes when we used more than 10 cells (data not shown). We re-