Manabu Hirata

Human pancreatic phospholipase A2 stimulates the growth of human pancreatic cancer cell line

Phospholipase A2 (PLA2) from human pancreas, designated hPLA2‐I, functions as a digestive enzyme. Interestingly, the present study demonstrated that the mature form of hPLA2‐I stimulated the growth of a human pancreatic cancer cell line MIAPaCa‐2, whereas the pro‐form was ineffective. PLA2s from Laticauda semifasciata fraction I, Crotalus adamanteus venom, Streptomyces violaceoruber and bee venom, showed no proliferative effect to the growth of MIAPaCa‐2. The Scatchard plot analysis revealed that the MIAPaCa‐2 cell had a specific binding site for the mature hPLA2‐I. The equilibrium binding constant (K d) and the maximum binding capacity (B max) were 2.6 nM and 0.4 fmol/106 cells, respectively. These results suggest that the mature hPLA2‐I, but not the pro‐form, may function as a growth factor of pancreas carcinoma via the specific binding site.

TP53 mutations in stage I gallbladder carcinoma with special attention to growth patterns

32 stage I cases of gallbladder carcinoma (GC) were examined to evaluate TP53 mutations with special attention to growth patterns. Their growth patterns were classified into two types: polypoid (P-type) and flat (F-type). 16 cases of GC were classified as P-type and 16 as F-type. p53 immunohistochemistry was performed using a mouse monoclonal anti-p53 antibody. Mutations in exons 5-8 were examined by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and direct sequencing. The incidence of p53 immunoreactivity was greater in the cases of F-type (11/16, 69%) than those in P-type (14/16, 25%) (P < 0.05). PCR-SSCP or direct sequencing revealed that TP53 mutations were detected in all cases positive for p53 protein. These results suggest that TP53 mutations may contribute to the carcinogenesis of the F-type GC, and than this pathway in the F-type may differ from that in the P-type GC.

Cholecystokinin receptor antagonist, loxiglumide, inhibits invasiveness of human pancreatic cancer cell lines

Recently, cholecystokinin has been reported to be important in regulating the growth of pancreatic cancer. We investigated the effect of loxiglumide (LXG), a cholecytskinin receptor antagonist, on the invasiveness of two human pancreatic cancer cell lines. Cells were treated with LXG for 24 h, and examined in the invasion assay. The expression and activity of MMP‐9 in supernatants from cancer cells were analyzed by Western blotting and zymogram. Interestingly, the invasiveness of cancer cells and expression of MMP‐9 were decreased by LXG in a dose‐dependent manner. LXG may be a useful therapeutic agent against pancreatic cancer.

Expression of the proliferative cell nuclear antigen (PCNA) in adenocarcinoma of the gallbladder, and its relationship to prognosis

Proliferative cell nuclear antigen (PCNA) has been correlated with degree of differentiation in some tumours, but information on PCNA expression in adenocarcinoma of the gallbladder is currently limited. Therefore, we examined PCNA expression in adenocarcinoma of the gallbladder, and its relationship to prognosis. The expression of PCNA was studied by immunohistochemistry in 70 formalin-fixed, paraffin-embedded specimens of surgically removed adenocarcinomas of the gallbladder. The percentage of stained nuclei was recorded, and the PCNA-labelling index (LI) was expressed as the ratio of labelled nuclei to the total number of nuclei counted. In all histological types, the PCNA-LI in the invasive zone of the tumour was higher than that in the luminal zone of the tumour (p < 0.05). The PCNA-LI showed a stepwise increase with decreasing degrees of differentiation in both the invasive and the luminal zone of the tumour (p < 0.01). In advanced adenocarcinomas, patients whose tumours had a PCNA-LI of less than 35 in the invasive zone had significantly longer survival rates than those with PCNA-LI equal to or greater than 35 (p < 0.01). Multivariate analysis, using the Cox proportional hazards model, indicated that a PCNA-LI > or = 35 in the invasive zone of the tumour was a significantly unfavourable prognostic factor (p = 0.002). The PCNA-LI of routinely processed specimens of adenocarcinoma of the gallbladder may be helpful for the evaluation of cell proliferation and prognosis.

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-

Comparative studies of epidermal growth factor, epidermal growth factor receptor, K-ras, and P53 in gallbladder carcinomas with and without an anomalous junction of pancreticobiliary duct

AIM: We reported a 6 to 10 times higher incidence of gallstone disease in curatively gastrectomized patients for cancer (Gastroenterology 104: A362, 1993) and a possible factor for the lithogenesis to be bile infection (Gastroenterology 106: A340, 1994). The aim of this study is to experimentally investigate whether bile infection and postgastrectomy gallstone disease is related with methods of gastrectomy for cancer, i.e., a conservative pylorus-preserving gastrectomy (PPG) and a radical Billroth II subtotal gastrectomy (B-II). METHOD: Five mongrel dogs underwent PPG with the truncal vagi and the hepatic branch intact and 6 dogs underwent B-I1 with wide bursectomy and bilateral truncal vagotomy. Other 6 dogs undergoing only laparotomy (sham operation) were served as the controls. The dogs were maintained with an ordinary laboratory canine diet for 1 year. Gallbladder (GB) bile was collected by sterile fine needle puncture during laparotomy at the time of surgery, 6 months and 12 months. The obtained bile was cultured for bacteriology and analyzed for chemical constituents. Bile acids were quantified by GLC. The presence or absence of gallstone (GS) was consecutively examined by ultrasound at 2 week intervals and by inspection of the aspirated bile. GSs were examined with infrared spectral analysis for classification. RESULTS: The PPG and control dogs incurred no bile infection nor gallstone disease. Three Of the 6 B-II dogs incurred bile infection (E. coli, Bacteroides and Enterococcus). GSs (black stones containing calcium bilirubinate) developed in 3 of the 6 B-II dogs (1 identified at 6 months and 2 at 12 months). Two of the 3 B-II dogs with GS were with bile infection but one without. Total bile acids and bile acid fractions did not significantly differ among the three animal groups. Free bile acids was detected in all of the B-II dogs and in 2 of the 5 PPG dogs but in none of the control dogs. Lithogenicity calculated was within the micellar zone for all dogs. CONCLUSION: Calcium bilirubinate stone developed only in the B-II gastrectomized dogs. Positive bacterial culture and detection of free bile acids were most common in the B-II dogs, which suggested that (1) bile infection is involved in postgastrectomy gallstone disease, (2) B-II subtotal gastrectomy is more liable to bile infection and gallstone disease and (3) PPG may preserve a better sphincter of Oddi motor function, thereby preventing retrograde biliary infection. COMPARATIVE STUDIES OF EPIDERMAL GROWTH FACTOR, EPIDERMAL GROWTH FACTOR RECEPTOR, K-RAS, AND P53 IN GALLBLADDER CARCINOMAS WITH AND WITHOUT AN ANOMALOUS JUNCTION OF PANCREATICOBILIARY DUCT. Keiji Hanada, Masaki Itoh, Kiyomu Fujii, Akira Tsuchida, Shouhei Ishimaru, Manabu Hirata, Toshiyasu Iwao, and Goro Kajiyama. First Dept. of Internal Medicine, Hiroshima University School of Medicine, Hiroshima, Japan. AIMS. Previously, we reported that mutations of Kras and p53 contributed to carcinogenesis in gallbladder mucosa with an anomalous junction of the pancreaticobiliary duct (AJPBD). In this study, we examined the expression of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR), and mutations of K-ras or p53in gallbladder carcinoma with and without AJPBD. METHODS. We used l0 gallbladder carcinomas with AJPBD (Group A), and 25 gallbladder carcinomas without AJPBD (Group B). EGF and EGFR were examined using immnohistochemistry. Genomic DNA was extracted from routinely processed tissues. Polymerase chain reaction (PCR) restriction fragment length polymorphism was performed for mutations of K-ras. Mutations of p53 were examined using PCR single strand conformation polymorphism. RESULTS. The percentage of immunoreactivity for EGF and EGFR in Group A (70%) was higher than that in Group B (40%). The incidence of mutation of Kras in Group A (30%) was higher than that in Group B (7%). As for p53, although mutations were found in only exon 7 and 8 in Group A, they were found in exon 5, 6, and 7 in Group B. CONCLUSIONS. i) The autocrine loop of EGF and EGFR may play an important role in tumor growth of gallbladder carcinomas associated with AJPBD. 2) Mutations of K-ras may strongly contribute to carcinogenesis of gallbladder carcinomas associated with AJPBD. 3) Mutation spots of p53 in gallbladder carcinomas associated with AJPBD may be different from that in gallbladder carcinomas without AJPBD.