Hepatocyte growth factor (HGF), a potent mitogen for adult rat hepatocytes in primary culture, has previously been shown to be primarily expressed in the nonparenchymal cells of the liver. Using polyclonal antisera against human and rat HGFs we studied the tissue distribution of HGF immunohistochemically and found the most intense staining in the pancreas islet cells in both man (autopsy cases) and the rat. Differential localization of 4 pancreas islet hormones, glucagon, insulin, somatostatin and pancreatic polypeptide, revealed HGF to be preferentially expressed within the glucagon‐positive cells. The results indicate that HGF is primarily produced or stored in A‐cells and may act as a growth factor in a paracrine and an endocrine fashion, like various other hormones.

The presence of ras family and p53 gene mutations in rat forestomach, intestine and liver tumors and soft tissue sarcomas induced by Nmethyl‐N‐nitrosourea (MNU) was examined using polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) followed by direct sequencing analysis. In the forestomach squamous cell carcinomas (SCC), Ha‐ros and p53 mutations were detected in 2 (40%) and 4 (80%) of 5 cases, respectively. The figures for Ki‐ras and p53 gene mutations in adenocarcinomas of the large and small intestines were 3 (18.8%) and 5 (31.3%) of 16 cases. Soft tissue sarcomas in different sites were found to have mutations of Ki‐ras in 7 (23.3%)and of p53 in 9 (30%) of 30 cases. One forestomach SCC and 2 soft tissue sarcomas had double p53 mutations in different exons. Single cases of forestomach SCC and intestinal adenocarcinoma had mutations in both Ki‐ras and p53 genes. No mutations were found in counterpart benign tumors or hepatocellular adenomas. The p53 mutation spectrum revealed preferential clustering within exon 8 for the forestomach SCCs, and exons 5 and 8 for the intestinal adenocarcinomas, whereas the distribution was evenly spread through exons 5 to 8 in soft tissue sarcomas. All the detected ras or p53 mutations were G:C to A:T transitions. These results indicate firstly that specific Ki‐ras, Ha‐ras and p53 gene mutations in MNU‐induced lesions are related to particular alkylation sites (G:C to A:T transitions) and secondly, although not essential, Ki‐ras, Ha‐ras or p53 gene mutations may be involved in the progression stage of forestomach, intestine and soft tissue neoplasms induced by MNU.

The expression of connexin 32 (Cx32), a major liver gap junction protein, after partial hepatectomy (PH) and during development and progression of hepatocarcinogenesis was studied in the rat. Cx32 was quantitatively analyzed by counting immunohistochemically demonstrated protein spots on the membranes of hepatocytes. Livers were sequentially examined after PH to assess the correlation with cell proliferation. For the analysis of different stages in carcinogenesis, Cx32 was assayed in N-ethyl-N-hydroxyethylnitrosamine-induced enzyme altered foci (EAF), hyperplastic nodules (HN), hepatocellular carcinomas (HCC), pulmonary metastatic HCC and transplanted HCC in relation to their degree of altered enzyme expression. Cx32 showed: (i) a rapid decrease after PH to its lowest levels during and 12 h after the S phase of cell proliferation when 5-bromo-2'-deoxyuridine (BrdU) labeling indices were examined; (ii) a progressive decrease from early preneoplasia EAF to HN and HCC, values for pulmonary metastatic and transplanted HCC being 0; (iii) clearly inverse correlations with increased BrdU index and degree of altered enzyme expression in HN, indicating that these, with the lowest Cx32 count, are closest to HCC. Therefore, the observed decrease appears linked to cell proliferation and progression of hepatocarcinogenesis, providing a reflection of cellular independence and growth advantage.

We analyzed clinical features and genetic alterations in the noncancerous and cancerous biliary lesions obtained from pancreaticobiliary maljunction (PBM) patients. Gallbladder (GB) and bile duct (BD) lesions were obtained surgically from 36 patients with PBM, and polymerase chain reaction (PCR) methods were used to examine for mutations of the K-ras gene and the p53 gene and for microsatellite instability (MSI). The 36 cases were clinically classified into two types according to whether extrahepatic bile duct dilatation was present: a congenital choledochal dilatation (CCD) group (n = 20) and a noncongenital choledochal dilatation (NCCD) group (n = 16). In the NCCD group, all 16 GB specimens exhibited hyperplastic, dysplastic, and cancerous (n = 9) lesions, but no pathological lesions were detected in the 12 BD specimens. On the other hand, in the CCD group, pathological examination revealed lesions, including 8 cancerous lesions, in 60% of the 20 GB specimens and lesions, and including 8 cancerous lesions, in 65% of the 20 BD specimens. K-ras mutations and MSI were detected in 33.3% and 0%, respectively, of 9 hyperplastic lesions, 28.6% and 85.7%, respectively, of 7 dysplastic lesions, and 60.0% and 80.0%, respectively, of 25 cancerous lesions (p <0.05; MSI in hyperplasia vs. dysplasia and cancer). There was no difference of the frequency in K-ras mutations and MSI between the NCCD and CCD groups. By contrast, p53 mutations were detected only in the cancerous GB lesions of both types, the rate being 35.3%. Genetic alterations of K-ras, MSI, and p53 are strongly associated with biliary tract cancer in PBM patients. MSI appears to contribute to carcinogenesis in the biliary tract mucosa of PBM patients, and p53 mutations may be related to the development of GB cancer in the CCD group.

The histogenesis of 3 types of rat renal cell tumors (basophilic cell, clear cell, and oncocytic) was stereologically analyzed, with particular attention paid to transitions from normal tubules. Early nitrosamine-induced preneoplastic lesions, including dysplastic tubules (altered tubules), epithelial hyperplasias, and small adenomas, were reconstructed using serially sectioned specimens processed for carbonic anhydrase type II (CA) and periodic acid-Schiff (PAS) (CA-PAS) double staining to allow easier distinction of the nephron segments: Proximal tubules had a PAS-positive brush border and were weakly positive for CA in the cytoplasm; distal tubules were PAS negative and weakly positive for CA; collecting ducts were PAS negative and strongly positive for CA. Similarly, cytochrome c oxidase (CytOx) and CytOx-PAS double staining was also applied to confirm the character of oncocytic lesions. All basophilic lesions (7 of 7) showed transition to proximal tubules. Clear cell lesions positive for CA, on the other hand, showed transition to distal tubules in 4 of 9 (44.4%) lesions and to collecting ducts in 4 of 9 (44.4%) lesions, but in only 1 of 9 (11%) to a proximal tubule. All oncocytic lesions (1,8,9,23,30,32) similar to those observed for lesions in the liver (6,11,12,16,25,32) and other organs (28,31). The histogenesis of experimentally induced renal tumors has been studied on the basis of characteristic features for enzyme-altered tubules considered to be early precursor lesions for renal cell tumors. For example, basophilic tubules with high glucose-6-phosphate dehydrogenase (G6PD) activity and decreased succinate dehydrogenase (SD) appear to arise from proximal tubules, whereas the oncocytic type exhibiting the opposite phenotype of low G6PD and high SD has been suggested to have a histogenesis independent from other segments, possibly collecting ducts (2,9,22,36 (27,29,34,35 cells (26,37). Similarly, cytochrome c oxidase (CytOx) immunostaining was used for the differential recognition of oncocytic lesions (17,19), occasionally in combination with PAS staining (CytOx-PAS). MATERIALS AND METHODSAnimals and Treatments. Groups of male 6-wk-old Fischer 344 (F344) and Sprague-Dawley rats (Charles River Japan, Inc., Atsugi, Japan) were administered 0.1 % Nethyl-N-hydroxyethylnitrosamine (EHEN) (Sakai Chemical Laboratories, Fukui, Japan) for 3 wk (5 rats) (18, 30, 32) and 0.07% N-nitrosomorpholine (NNM) (Nakalai Tesque, Inc., Kyoto, Japan), respectively, mixed in their drinking water for 10 wk (5 rats) (9) and were then returned to tap water. They were then placed on basal diet (Oriental MF, Oriental Yeast Co., Ltd., Tokyo, Japan) throughout the experimental period. Animals were sacrificed 30 wk after the commencement of carcinogen application. Resected kidneys were cut into 3.5-to 4-mmthick slices along the long axis with a razor blade, fixed in ice-cold acetone for 4 wk, and then routinely processed for paraffin embedding.Histological Preparations and Stereological Reconstruction. Paraffin-embedded kidneys, ...