Hirofumi Suwa

Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas.

It has been reported that the rho genes, which consist of a ras-related small GTPase protein family, regulate cytoskeletal structures and have the potential to transform cultured cells. To investigate the biological relevance of the rho genes in pancreatic carcinogenesis, we examined expressions of the rhoA, B and C genes by polymerase chain reaction after reverse transcription (RT-PCR) in 33 cases of ductal adenocarcinoma of the pancreas. In addition, mutations of the K-ras, rhoA, B and C genes were studied in the same series of tumour tissues to correlate with rho gene expressions. The expression levels of the rhoC gene were significantly higher in tumours than in non-malignant portions (P < 0.001). Metastatic lesions overexpressed the rhoC gene compared with primary tumours (P < 0.05). Carcinoma tissues with perineural invasion and lymph node metastasis exhibited significantly higher expressions of the rhoC gene than tumours without these manifestations (P < 0.001 and P < 0.05 respectively). Overexpression of the rhoC gene significantly correlated with poorer prognosis of patients with pancreatic adenocarcinoma (P < 0.05). In contrast, the expression levels of the rhoA and B genes showed no significant relationship with clinicopathological findings. Mutation was not found either in the rhoA, B or C gene sequences examined. K-ras gene mutation, detected in 27 out of 33 (81.8%) cases, did not affect the expression levels in any of the rho genes. These suggest that elevated expression of the rhoC gene may be involved in the progression of pancreatic carcinoma independent of K-ras gene activation.

Low Incidence of Point Mutations in H- ,K- and N-ras Oncogenes and p53 Tumor Suppressor Gene in Renal Cell Carcinoma and Peritoneal Mesothelioma of Wistar Rats Induced by Ferric Nitrilotriacetate

An iron chelate, ferric nitrilotriacetate (Fe‐NTA), induces renal proximal tubular damage, a consequence of iron‐catalyzed free radical reactions, that finally leads to a high incidence of renal cell carcinoma (RCC) in rodents. Previous studies have identified, within 24 h after administration of Fe‐NTA, lipid peroxidation products, aldehyde‐modified proteins and a variety of modified DNA bases such as 8‐hydroxyguanine that may be mutagenic in vivo. In the present study, pathological features of the RCCs were studied, and, in an effort to correlate them with carcinogen‐specific molecular events in Fe‐NTA‐induced carcinogenesis, the H‐, K‐ and N‐ras oncogenes and the p53 tumor suppressor gene were investigated for the presence of mutations. Fe‐NTA‐induced RCCs showed similarity to human RCCs in that they are often invasive, metastatic and fatal. None (0 of 12) of the tumors had mutation in codons 12, 13 and 61 of the H‐, K‐ and N‐ras genes by direct sequencing. Only one (1 of 12) tumor with high grade histology revealed a CGC‐to‐CTC (Arg to Leu) transversion in codon 246 of the p53 gene by the use of single strand conformation polymorphism (SSCP) analysis and direct sequencing. High expression of mutant p53 protein was confirmed by Western blotting and immunohistochemistry. Study of three peritoneal mesotheliomas induced by Fe‐NTA revealed no mutation in ras and p53 genes. These results suggest that the ras and p53 genes are not the major targets of mutation in Fe‐NTA‐induced carcinogenesis of kidney and mesothelium. Instead, p53 mutation may work for potentiation of malignant character in Fe‐NTA‐induced renal carcinogenesis.

Immunohistochemical study of metallothionein in pancreatic carcinomas

Metallothioneins are a family of intracellular metalloproteins that have been thought to be involved in anticancer drug resistance. However, the role of metallothioneins in pancreatic cancer has not been investigated in detail The immunohistochemical localization of metallothionein was examined in normal human adult pancreas tissue and in 75 pancreatic duct cell carcinomas, using monoclonal anti-metallothionein antibody. Furthermore, in vitro studies on the sensitivity of pancreatic cancer to cisplatin were performed in 10 cases of pancreatic carcinoma. Metallothionein staining was wekly positive in the acinar and islet cells and intralobular ducts but was negative in the large pancreatic ducts. In pancreatic carcinomas, metallothionein staining was diffusely positive in 6 (8%), focally positive in 25 (33%) and negative in 44 (59%) of the 75 pancreatic carcinomas. The expression of metallothioneins in pancreatic tumors was related to metastasis, poor prognosis and poor histological grading (poorer glandular differentiation and nuclear anaplasia). The in vitro study of tumor sensitivity to cisplatin showed no significant correlation between metallothionein expression and resistance to cisplatin. Metallothionein-positive pancreatic carcinoma will be potentially highly malignant or acquire an enhanced ability to produce metallothioneins as the malignant potential increases. The expression of metallothionein could be a prognostic indicator in pancreatic carcinomas.

Quantitative Analysis of Collagen and Collagen Subtypes I, III, and V in Human Pancreatic Cancer, Tumor-Associated Chronic Pancreatitis, and Alcoholic Chronic Pancreatitis

The collagen content in human pancreatic cancer tissue, tissue of tumor-associated chronic pancreatitis (TACP), and normal pancreatic tissue was determined in 14 patients with pancreatic cancer by measuring the amount of 4-hydroxyproline. Four patients with alcoholic chronic pancreatitis (AlCP) were also analyzed. The mean collagen content in both pancreatic cancer tissue and TACP tissue was approximately threefold higher than in normal pancreatic tissue. Cyanogen bromide peptides of type I, III, and V collagens from invasive ductal carcinomatous tissue of the pancreas and from TACP tissue of eight patients were analyzed sequentially using high-performance liquid chromatography with ion-exchange and gel-permeation columns. No difference in the proportion of type I, 111, and V collagens was detected between pancreatic cancer tissue and TACP tissue. The mean collagen content in AlCP tissue was significantly lower than that in TACP tissue, but no difference in the proportion of type I, 111, and V collagens was detected between these two tissues. These results indicate a similar quantity and distribution pattern of fibrillar collagen in human pancreatic cancer and TACP.

Clinical significance of serum p53 antigen in patients with pancreatic carcinomas.

BACKGROUND: Alterations in the p53 gene are often found in pancreatic cancer, and accumulation of the p53 protein has been noted in tumour cells. AIMS: To investigate whether serum p53 protein concentrations could be used as markers for p53 gene mutations in neoplasms of the pancreas. METHODS: Serum p53 protein concentrations were determined by an enzyme linked immunosorbent assay (ELISA) in 104 cases of pancreatic adenocarcinoma, and 61 matched formalin fixed tissue sections were also stained by an anti-p53 DO-7 monoclonal antibody. RESULTS: The mean serum concentration of p53 protein in the adenocarcinoma patients was 0.27 (SEM 0.02) ng/ml, and was significantly higher than in 35 healthy blood donors (0.15 (0.02) ng/ml, SD = 0.11) or in 15 cases of chronic pancreatitis (0.15 (0.02) ng/ml). Adopting an arbitrary cut off value for the serum p53 protein concentration of 0.37 ng/ml, which corresponded to a value 2 SD above the mean value from the healthy blood donors, positive serum p53 protein concentrations were found in 23 out of 104 (22.1%) patients with adenocarcinomas examined, 16 out of 47 (34.0%) patients with carcinomas with distant metastases, but only seven of 57 patients (12.3%) with carcinomas without metastases (p < 0.05). In 11 patients with pancreatic adenocarcinomas, the mean serum p53 protein concentration after tumour resection was 0.21 (0.05) ng/ml, and had decreased compared with the preoperative concentrations (0.25 (0.05) ng/ml) (P < 0.05). There were no significant associations between the serum concentrations of p53 protein and serum concentrations of markers such as CA19-9 or CEA; however, serum concentrations of p53 protein demonstrated a potential role as an additional tumour marker. Immunohistochemical studies disclosed that the p53 protein was expressed in 28 out of 61 pancreatic adenocarcinomas (45.9%). Serum p53 protein concentrations in the positively immunostained cases were significantly higher than in the negatively immunostained cases (0.35 (0.05) ng/ml v 0.15 (0.01) ng/ml; p < 0.005). Furthermore, positive immunostaining for p53 protein was found in eight out of 10 (80%) serum positive p53 protein cases with adenocarcinomas. CONCLUSION: An increase in serum p53 protein concentrations appears during the progression of pancreatic adenocarcinoma and correlates with the accumulation of p53 protein as a result of a mutation of the p53 gene. An analysis of p53 antigen concentrations can detect p53 gene alterations, which could be useful for the selection of treatment regimens.

Immunohistochemical localization of P-glycoprotein and expression of the multidrug resistance-1 gene in human pancreatic cancer : Relevance to indicator of better prognosis

We investigated immunohistochemical localization of P‐glycoprotein (F‐gp) on paraffin‐embedded sections from 103 cases of previously untreated pancreatic tumors and also analyzed multidrug resistance‐1 (MDR1) gene expression by polymerase chain reaction after reverse transcription in 35 cases. High positive staining for P‐gp was observed in 72.8% of pancreatic tumors and in 73.2% of ductal adenocarcinoma. In ductal adenocarcinoma, immunoreactivity of P‐gp was inversely correlated with biological aggressiveness of tumors determined by histologic grading (P<0.01), tumor size (P<0.01), retroperitoneal invasion (P<0.01) and portal invasion (P<0.05). Expression of the MDR1 gene was detected in all the pancreatic tumors examined and was significantly higher than that in normal pancreas (P<0.05). The levels of MDR1 mRNA showed a moderate correlation with those of P‐gp (r=0.62, P<0.0001). Higher expression levels of MDR1/P‐gp significantly correlated with better prognosis of patients with ductal carcinoma (P<0.05). Among patients with ductal carcinoma, the high staining group for P‐gp revealed a 3.5‐fold better prognosis compared with the low staining group (HR=3.47, 95% CI=1.62, 7.45; P=0.0016). In conclusion, MDR1 gene/P‐gp expression in pancreatic cancer without chemotherapy inversely correlates with biological aggressiveness and is an independent indicator of favorable prognosis.

Expression of membrane-type matrix metalloproteinase-1 in human pancreatic adenocarcinomas

Abstract The expression of a new type of matrix metalloproteinase, membrane-type matrix metalloproteinase-1 (MT-MMP-1), was examined in 24 cases of primary pancreatic adenocarcinomas and 9 cases of secondary liver tumors derived from pancreatic adenocarcinomas, using a non-radioactive in situ hybridization and immunohistochemical methods. Out of 24 cases of primary pancreatic adenocarcinomas, 18 showed positive expression of MT-MMP-1 transcripts in cancer cells and 20 of 24 showed positive expression in the tumor stromal cells. The immunoreactivity of the gene products for MT-MMP-1 was demonstrated to be almost the same, as shown by in situ hybridization in these 24 cases. In particular, both the staining intensity for MT-MMP-1 transcripts and the immunoreactivity of the gene products in the tumor stromal cells of mucinous cystadenocarcinomas were significantly weaker than those of common-type ductal adenocarcinomas among the 24 cases. All of the 9 cases of secondary liver tumors derived from pancreatic adenocarcinomas showed positive expression for MT-MMP-1 transcripts but less immunoreactivity for the gene products. These results suggest that MT-MMP-1 is transcribed and translated in both cancer cells and the tumor stromal cells in human pancreatic adenocarcinomas. Furthermore, considering that common-type ductal adenocarcinoma of the pancreas usually shows a strong desmoplastic reaction, while mucinous cystadenocarcinoma typically does not, MT-MMP-1 expressed in the tumor stromal cells of common-type adenocarcinomas may be involved in processes leading to the desmoplastic reaction.

Evaluation of cholecystokinin, gastrin, CCK-A receptor, and CCK-B/gastrin receptor gene expressions in gastric cancer

The brain-gut hormones, cholecystokinin (CCK) and gastrin, regulate the growth of gastrointestinal mucosa and tumor cells. In this study, reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate messenger RNA expression for CCK, gastrin, CCK-A receptor, and CCK-B/gastrin receptor in surgical specimens of gastric cancers and in normal antrum and body mucosa of the stomach. The CCK mRNA expression was detectable in 4/14 (29%) samples of gastric cancer and in 3/12 (25%) samples of antral mucosa. However, the gastrin mRNA expression was not detectable in any gastric cancer samples, although it was detectable in all the samples of antral mucosa. The CCK-A receptor mRNA expression was detectable in 5/14 (36%) samples of gastric cancer and in 7/12 (58%) body mucosa. Three cases out of 14 (21%) of gastric cancer expressed both CCK gene and CCK-A receptor gene. The CCK-B receptor mRNA expression was detectable in only 1/14 (7%) samples of gastric cancer, although it was detectable in 10/12 (83%) body mucosa of the stomach. These findings may suggest a greater role for CCK and CCK-A receptor than for gastrin and CCK-B receptor in gastric cancers.

K-ras and p53 Alterations in Genomic DNA and Transcripts of Human Pancreatic Adenocarcinoma Cell Lines

We analyzed 15 human pancreatic adenocarcinoma cell lines for alterations of the K‐ras and the p53 genes and their transcripts. In 11 cell lines (73.3%), point mutations of the K‐ras gene were found at codon 12 in exon 1. In 9 cell lines one allele was mutated and the other was wild type, and both the alleles were expressed into mRNA. In one cell line both alleles of codon 12 were mutated to TGT and GTT, respectively, hut only TGT was transcribed into mRNA. Alterations in mRNA of the p53 gene were detected in 10 cell lines (66.7%). Analysis of the genomlc sequence of the p53 gene revealed that the alterations consisted of 6 cases of base pair substitutions and 1 case of 1‐bp deletion in evolutionarily conserved exons 5 to 8, 2 cases of splicing mutations in exon 4, and 1 case of novel deletion from exons 2 to 9. In 14 cell lines (93.3%), alterations were identified in the K‐ras or p53 gene. Of these, 4 cell lines harbored K‐ras mutations without p53 alteration, whereas 3 cell lines exhibited p53 alterations without K‐ras mutation. Thus, it is suggested that activation of the K‐ras gene and inactivation of the p53 gene are strongly and cooperatively associated with pancreatic carcinogenesis.

DISTRIBUTION OF THE CARBOHYDRATE ANTIGENS, DU-PAN-2 AND CA19-9, IN TUMORS OF THE LUNG

Aims and background The carbohydrate chains of malignant cells appear to be related to oncofetal differentiation. The serum levels of CA19-9 have been reported to be evaluated in some patients with lung carcinomas, however, the distribution of carbohydrate antigens were not precisely described. We have investigated in this study the distribution of DU-PAN-2 and CA19-9 antigens in lung tumors. Methods Ninety five specimens of lung tumors were selected from surgical specimens. The expression of DU-PAN-2 and CA19-9 were studied by immunohystochemical techniques. The relationship between the expression of these antigens and the classification or the differentiation degree of the tumors were examined. Results DU-PAN-2 or CA19-9 antigens were detected in 41 (54%) and 45 (59%) cases of the 76 malignant epithelial tumors investigated. These antigens were detected in all types of malignant epithelial tumors, including squamous cell carcinomas, where they were mainly localized to the entire cell surface of malignant cells. In adenocarcinomas, large cell carcinomas and small cell carcinomas, however, these antigens were commonly detected both on the cell membrane and in the cytoplasm. There was positive correlation between the degree of differentiation and DU-PAN-2, but not CA19-9 expression. Among the non-epithelial tumors investigated, those antigens were detected in pulmonary blastomas but not in mesotheliomas. Conclusions Immunohistochemical studies for DU-PAN-2 and CA19-9 are useful for defining characteristics of the lung tumors. DU-PAN-2 could be a marker for differentiating between malignant epithelial tumors and mesotheliomas.