Hideo Shimura

HGF/NK4 is a specific antagonist for pleiotrophic actions of hepatocyte growth factor

We prepared a specific antagonist for hepatocyte growth factor (HGF) and designated it HGF/NK4. HGF/NK4 is composed of N‐terminal 447 amino acids of the α‐chain of HGF, thus contains the N‐terminal hairpin domain and subsequent four kringle domains. HGF/NK4 competitively inhibited the specific binding of HGF to the receptor. Importantly, HGF/NK4 neither stimulated DNA synthesis of primary cultured rat hepatocytes (mitogenesis) nor induced cell scattering (motogenesis) and branching tubulogenesis (morphogenesis) of MDCK renal epithelial cells, however, HGF/NK4 almost completely inhibited the mitogenic, motogenic, and morphogenic activities of HGF. HGF/NK4 also suppressed tyrosine phosphorylation of the c‐Met/HGF receptor induced by HGF. Apparently this is the first documentation of a specific antagonist which abrogates the mitogenic, motogenic, and morphogenic activities of HGF.

Acquisition of invasive phenotype in gallbladder cancer cells via mutual interaction of stromal fibroblasts and cancer cells as mediated by hepatocyte growth factor

Growth and motility of carcinoma cells are regulated through their interactions with host stromal cells, i. e., tumor‐stromal interactions. Hepatocyte growth factor (HGF), a ligand for c‐Met tyrosine kinase, is a stromal‐derived regulator of growth, motility, and morphogenesis. HGF stimulated proliferation and motility of GB‐d1 gallbladder carcinoma cells from a patient with gallbladder cancer. HGF induced in vitro invasion of GB‐d1 cells into a collagen gel matrix, and this potent, invasive effect was not seen with epidermal growth factor, transforming growth factor‐β1, basic fibroblast growth factor, or platelet‐derived growth factor. Although GB‐d1 did not produce HGF, the cells did produce a factor which enhances HGF production in human skin fibroblasts, and this factor proved to be interleuldn‐1β (IL‐1β). When GB‐d1 cells were co‐cultured with fibroblasts such that a collagen gel matrix was layered between the GB‐d1 cells and fibroblasts, GB‐d1 cells invaded the gel, but invasion of the cells in the co‐culture system was inhibited by antibodies against HGF and partially inhibited by antibodies against IL‐1β. Thus, GB‐d1 cell‐derived IL‐1β stimulates HGF production in stromal fibroblasts and HGF up‐regulated in the fibroblasts induces invasion of GB‐d1 cells. The looped interaction of carcinoma cells and stromal fibroblasts mediated by HGF and a HGF‐inducer such as IL‐1β may be one mechanism which would explain the acquisition of malignant phenotype through tumor‐stromal interactions.

Laparoscopic treatment of congenital choledochal cyst

Abstract. We describe the laparoscopic treatment of a patient presenting with congenital choledochal cyst. Our patient was a 19-year-old man with a complaint of recurrent abdominal pain due to pancreatitis. The choledochal cyst was type I and had a common channel of pancreatobiliary duct, as revealed by endoscopic retrograde cholangiopancreatography. Under laparoscopic guidance, the dilated bile duct and the gallbladder were excised, and a Roux-en-Y anastomosis was constructed with an endo-EEA. Finally, end-to-side anastomosis was carried out by the continuous suture method, aided by an Endostitch between the stump of the hepatic duct and the Roux-en-Y limb. After the operation, slight hyperamylasemia was observed for several days but further treatment was not necessary. Postoperative symptoms were minimal, and the patient was discharged on the 11th day after the procedure. Although it is difficult and time-consuming, laparoscopic operation is highly beneficial for the patient. The use of such instruments as the endostapler and Endostitch may help to simplify this complex intracorporeal procedure involving division and anastomosis of the digestive tract.

Effects of inhibitors of the cytoplasmic structures and functions on the early phase of infection of cultured cells with simian virus 40

To obtain information about cytoplasmic structures and functions involving the entry of simian virus 40 virions into cells, we examined whether the inhibitors that affect the functions and/or structure of lysosomes, cell membrane, and cytoskeletons inhibit expression of nuclear T antigen in the SV40-inoculated rat 3Y1 and monkey CV-1 cells. Chloroquine, methylamine, and butylamine did not inhibit T-antigen expression, suggesting that lysosomal acidification is not required for establishment of infection. Cytochalasin B had no effect, suggesting that microfilaments are not involved. Monensin, colcemid, and amantadine each inhibited T-antigen expression at doses causing no obvious cytotoxicity. Maximal inhibition was seen when these inhibitors were added to the cultures within 1 hr (monensin), within 4 hr (colcemid), or within 12 hr (amantadine) after virion adsorption to the cell surface. When the inhibitor was present in the virus-inoculated cultures for 24 hr and then removed, nuclear T antigen began to be expressed at 4 hr (monensin), 9 hr (colcemid), or 1 hr (amantadine) after removal of the inhibitors. Results of SDS-PAGE analysis of immunoprecipitated radiolabeled proteins of infected cells revealed that amantadine inhibited synthesis of large and small T antigens as well as general protein synthesis. Inhibition by colcemid may be due to disruption of microtubules, because other microtubule-disrupting agents (colchicine, vinblastine, nocodazole, and podophyllotoxin) also inhibited appearance of nuclear T antigen but lumicolchicine and taxol did not. Electron microscopy revealed that, in the presence of colcemid, although the adsorbed virions were readily internalized to form pinosomes, vectorial movement of the pinosomes to the nucleus appeared to be inhibited. Results of electron microscopy also suggest that inhibition by monensin may occur mainly in internalization of adsorbed virions and that the inhibition is leaky such that the early steps of infection proceed slowly in the presence of monensin. We conclude that monensin, colcemid, and amantadine interfere with mutually different early events of SV40 infection.

Hepatocyte growth factor stimulates the invasion of gallbladder carcinoma cell lines in vitro.

Human gallbladder cancer is highly malignant and its prognosis is usually poor depending on the extent of surrounding tissue invasion. We examined in vitro the invasive activity of four gallbladder cancer cell lines (GB-d1, GB-h3, GB-d2 and FU-GBC-1) in the absence or presence of hepatocyte growth factor (HGF). In type 1 collagen gel culture, HGF stimulated cell proliferation and induced an invasive phenotype of arborizing structures in GB-d1, GB-h3 and GB-d2. In a Matrigel invasion assay, invasion was also induced in three of these cell lines by HGF but not in FU-GBC-1. Cellular motility was, however, stimulated by HGF in all of the four cell lines to various extents. Zymography for proteolytic enzymes demonstrated high levels of type IV collagenase and urokinase-type plasminogen activator (u-PA) activity in GB-d1, GB-h3 and GB-d2 even in the absence of HGF. In the presence of HGF, the 72 kDa type IV collagenase (MMP-2) activity of GB-h3 and u-PA activities of GB-d1, GB-h3 and GB-d2 were enhanced. In contrast, the MMPs and PAs activities of FU-GBC-1 were faint irrespective of the addition of HGF. A Western blot analysis demonstrated higher levels of 190 kDa c-MET product (HGF receptor) of GB-d1, GB-h3 and GB-d2 than that of FU-GBC-1. The invasion in the Matrigel assay stimulated by HGF was inhibited by protease inhibitors, aprotinin and FOY-305, as well as by anti-HGF antibody. These results thus suggest that, in addition to the importance of the proteolytic activity, the cellular motility induced via the HGF/HGF-receptor system is essential for the invasive progression of gallbladder carcinoma cells.

Semi-quantitative analysis of telomerase in pancreatic ductal adenocarcinoma

Using a polymerase chain reaction-based amplification assay, we measured telomerase activity in surgically resected pancreatic ductal carcinomas (n=16 cases) and normal ducts (n=6), comparing findings with the telomerase activity of a human pancreatic cancer cell line, MIA PaCa-2, as a standard, i.e., relative telomerase activity was determined. Telomerase activity was expressed as the equivalent telomerase intensity of the number of cells of MIA PaCa-2 per μg protein of tissue samples. The median value for telomerase activity in normal pancreatic ducts was 0.13 and the 25th and 75th percentile were 0.01 and 0.76. The median value for telomerase activity in pancreatic ductal adenocarcinoma was 34.7 (25th percentile, 4.98; and 75th percentile, 296), significantly higher than that of normal ducts (P<0.001). When the cut-off value was set at 1.0 and 3.0, the telomerase positivity rate of pancreatic ductal adenocarcinomas was 100% and 81.3%, respectively. Telomerase may be a specific marker for pancreatic ductal carcinomas.

Laparoscopic treatment of duodenal carcinoid tumor. Wedge resection of the duodenal bulb under endoscopic control.

Abstract. A 46-year-old man with epigastralgia and slight elevation of urinary 5-hydroxyindole acetic acid (5HIAA) was found to have a well-demarcated carcinoid tumor in the duodenal bulb. The tumor measured 8 mm in size, and showed submucosal involvement but no metastasis to the liver and regional lymph nodes. After laparoscopic exposure and lifting of the duodenal wall around the tumor, wedge resection of the duodenal bulb including the tumor was performed successfully with a laparoscopic endostapler under direct endoscopic control. The postoperative course of the patient was uneventful. Laparoscopic wedge resection of the duodenum would be an appropriate minimally invasive treatment for selected duodenal neoplasms with special preoperative assessments and intraoperative considerations.

Abortive transformation of rat 3Y1 cells by simian virus 40: Viral function overcoming inhibition of cellular proliferation under various conditions of culture

Resting cultures of nonpermissive rat 3Y1 cells were infected with simian and T antigen expression and entry into S phase were examined under various conditions of culture. In the complete absence of serum from the medium or at an extremely high cell density, the cells delayed T antigen expression and entry into S phase, leaving the interval between the two events constant. Results using the viral mutants deleted in the coding region for the small t antigen ruled out the role of this antigen in induction of S phase. From these and other results presented, we conclude that the large T antigen induces S phase with the same efficiency under different conditions of cultures. We also present the evidence that the large T antigen function is required and is sufficient for entry into S phase in the second as well as in the first generation.

Phospholipid liposomes enhance the infectivity of purified simian virus 40 virions

When simian virus 40 virions purified after treatment with sodium deoxycholate were incubated with the extract of monkey kidney CV-1 cells, infectivity of the virions was enhanced. The infectivity-enhancing activity was recovered from the phospholipid fraction of CV-1 cells. The constructed liposomes composed of phosphatidylserine were able to enhance the infectivity of the purified virions, but those composed either of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, or phosphatidylinositol could not. The liposomes constructed with a mixture of phosphatidylethanolamine and phosphatidylcholine at a ratio of 1:1 (w:w) also enhanced the infectivity of the purified virions. Pretreatment of cells with liposomes either of phosphatidylserine or of phosphatidylethanolamine did not enhance susceptibility of the cells to infection with the purified virions. These observations suggest that the major phospholipids of the cellular membrane, when associated with virions, play a vital role in activation of purified virions.

Decline in infectivity of simian virus 40 by sodium deoxycholate and its restoration with the extract of monkey kidney cells

Plaque-forming activity and T-antigen-synthesizing activity in the crude preparation of simian virus 40 (SV40) decreased to 1/20-27 after treatment with 0.5% sodium deoxycholate (DOC) for 30 min at 37 degrees C. A full restoration of the activity occurred after incubation of DOC-treated virions with the extract of monkey CV-1 cells, host cells for productive infection with SV40. Analysis by sedimentation through 15% sucrose to CsCl cushion (rho = 1.327 g/cm3) revealed that virions in the [35S]methionine-labeled crude virus preparation sedimented to the interface between CsCl and sucrose, and that treatment with DOC resulted in the loss of infectivity and the appearance of virions sedimentable into CsCl cushion. The [35S]methionine-labeled purified virions (prepared after treatment with DOC and sedimentable into CsCl cushion) sedimented to the CsCl-sucrose interface after incubation with the cell extract, with restoration of infectivity. The infectivity-restoring activity of the cell extract was sensitive to ethyl ether, partially sensitive to heating at 75 degrees-97 degrees for 30 min, but resistant to treatment with DNase (50 micrograms/ml), RNase (40 micrograms/ml), or trypsin (0.05%) for 30 min at 37 degrees. These results suggest that lipid-related cellular components bind stably to virions of SV40 and facilitate an efficient infection.