Overexpression of midkine in pancreatic duct adenocarcinomas induced by N-Nitrosobis(2-oxopropyl)amine in hamsters and their cell lines.

The expression of midkine (MK) was investigated in pancreatic ductal hyperplasias, atypical hyperplasias and adenocarcinomas induced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters, and in hamster ductal adenocarcinoma cell lines (HPD-1NR, -2NR and -3NR). MK mRNA was clearly overexpressed in invasive pancreatic duct adenocarcinomas (PCs) and the three cell lines as assessed by northern blot analysis, and MK protein expression increased from ductal hyperplasia through atypical hyperplasias, intraductal carcinomas and invasive PCs by immunohistochemistry. The extent of overexpression of MK mRNA in PCs was almost the same as in hamster whole embryonic tissue. MK is reported to be a retinoid-responsive gene, but MK mRNA expression was not affected by treatment with all-trans retinoic acid (tRA) or N-(4-hydroxyphenyl)retinamide (4-HPR) in HPD-1NR cells. The results thus suggest that MK expression is involved in the development and progression of pancreatic ductal adenocarcinomas induced by BOP in hamsters, with loss of upregulation by retinoic acid.     

A cyclooxygenase-2 inhibitor,nimesulide, inhibits postinitiation phase of N-nitrosobis(2-oxopropyl)amine-induced pancreatic carcinogenesis in hamsters

The modification effects of nimesulide, a cyclooxygenase (COX)-2 inhibitor, administration during the postinitiation phase of pancreatic carcinogenesis were investigated in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Male Syrian hamsters were given 4 weekly s.c. injections of BOP at a dose of 10 mg/kg and thereafter administered 0, 100 or 400 ppm nimesulide in the diet for 36 weeks. Additional groups of hamsters were fed 400 ppm nimesulide without prior BOP initiation or nontreated. At week 40, all surviving animals were killed and development of neoplastic and preneoplastic lesions was assessed histopathologically. The incidence of pancreatic adenocarcinomas was significantly (p < 0.05) decreased in the BOP/400 ppm nimesulide group compared to the BOP alone group. The multiplicity of total lesions of pancreatic adenocarcinoma plus atypical hyperplasia was also significantly (p < 0.05) lowered. Immunohistochemically, COX-2 was clearly expressed in pancreatic and lung tumor cells, whereas expression was not remarkably affected by the 400 ppm nimesulide treatment. Proliferating cell nuclear antigen labeling indices of pancreatic ducts were significantly (p < 0.01) reduced by nimesulide. The incidence and multiplicity of neoplastic lesions in other organs did not significantly differ among the BOP-treated groups, though only the multiplicity of lung tumors showed a tendency to decrease. No neoplastic lesions were detected in animals receiving nimesulide alone. Our results clearly indicate that nimesulide protects against BOP-induced pancreatic tumors in hamsters.     

Pancreatic ductal adenocarcinomas induced in Syrian hamsters by N-nitrosobis(2-oxopropyl)amine contain a c-Ki-ras oncogene with a point-mutated codon 12

We have used the polymerase chain reaction and dideoxynucleotide sequencing to amplify and sequence exons 1 and 2 of the c-Ki-ras proto-oncogene from the normal Syrian golden hamster. Similar methods were employed to screen for the presence of point mutations in the c-Ki-ras oncogene in primary hamster pancreatic ductal adenocarcinomas (PDC) induced by N-nitrosobis(2-oxopropyl)amine (BOP). A GGT to GAT point mutation was detected in codon 12 of the c-Ki-ras gene in 10 primary hamster PDCs. This same point mutation was present in two nonclonal cell lines, PC-1 and PC-1-0, established from tumors that were produced in hamsters by subcutaneously implanting a preparation of minced BOP-induced PDC. Two clonal cell lines, Cl-3 and Cl-7, were cloned from the PC-1 cell line, and these cell lines also carried the GAT point mutation at codon 12. This point mutation was the same as that detected in greater than 75% of adenocarcinomas from the human exocrine pancreas. Thus, our findings provide further validation for the use of the BOP-induced hamster PDC model as a relevant experimental model for human pancreas cancer: not only did the hamster pancreatic ductal adenocarcinomas closely resemble their human counterpart in histopathological morphology and sequential development, but they also contained the same point mutation in codon 12 of the c-Ki-ras oncogene, as has been reported for human pancreatic adenocarcinomas.     

Inhibition by methionine of pancreatic carcinogenesis in hamsters after initiation with N-nitrosobis(2-oxopropyl) amine

The modifying effects of dietary L-methionine in the post-initiation phase of pancreatic carcinogenesis were investigated in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Groups consisting of 20 and 30 animals, respectively, were given BOP subcutaneously, once a week five times at a dose of 10 mg/kg body wt. and then continuously fed diet supplemented with 2% (group 1) or 0% (group 2) methionine (weeks 5-32). After five subcutaneous injections of saline, group 3 animals were similarly fed diet supplemented with 2% methionine for the same period. The incidence of pancreatic ductal adenocarcinomas was significantly lower in group 1 (36.8%, P<0.05) than in group 2 (71.4%). Multiplicity of adenocarcinomas was also significantly lowered (0.52 and 1.28/hamster, P<0.05). Similarly, total numbers of combined adenocarcinomas and dysplastic lesions were significantly decreased in group 1 (2.05, P<0.05) as compared with group 2 (3.67). Methionine enhanced atrophic change of pancreatic acinar cells in hamsters given BOP, indicating that the inhibitory effects on the post-initiation stage of BOP-induced pancreatic carcinogenesis in hamsters could be generally linked to suppression of growth.     

Effects of catechol and its analogs on pancreatic carcinogenesis initiated by N-nitrosobis(2-oxopropyl)amine in Syrian hamsters

The modifying effects of the phenolic antioxidant catechol (CC) and its analogs hydroquinone (HQ) and resorcinol (RN) on pancreatic carcinogenesis were evaluated in 146 female Syrian golden hamsters. Groups of animals received either saline or 70 mg/kg body wt N-nitrosobis(2-oxopropyl)amine (BOP) s.c. injections, twice with a 2 week interval, followed by basal diet or diet containing 1.5% of CC, HQ or RN, and 0.75% CC from week 4. All hamsters were killed at week 20 and histopathologically examined for development of pancreatic, liver and gall bladder lesions. The total numbers of pancreatic lesions comprising carcinomas, atypical ductal hyperplasias and ductal hyperplasias per hamster were significantly decreased in animals receiving BOP followed by CC, HQ and RN when compared to those in hamsters given BOP followed by basal diet. Incidence values for atypical ductal hyperplasias were also significantly decreased by the RN or 0.75% CC treatments. The results thus suggest that pancreatic carcinogenesis initiated by BOP in Syrian hamsters can be inhibited by treatments with phenolic antioxidants such as CC, HQ and RN for a relatively short experimental period.     

Mechanism of prevention of development of N-nitrosobis(2-oxopropyl)amine induced pancreatic carcinoma by a protein-free diet in hamsters.

It has been shown that feeding a protein-free diet prior to injection of N-nitrosobis(2-oxopropyl)amine (BOP) can inhibit the development of pancreatic carcinoma in the hamster. The purpose of this study was to determine whether this inhibition is due to a direct effect on the pancreas or to a systemic mechanism. Pancreas transplantation was used to create four groups of two-pancreas hamsters (Group 1: protein-containing diet donor/protein-containing diet host; Group 2: protein-containing diet donor/protein-free diet host; Group 3: protein-free diet donor/protein-containing diet host; Group 4: protein-free diet donor/protein-free diet host). Animals were fed either a protein-containing diet or a protein-free diet for four weeks preoperatively. One day after transplantation, the two-pancreas animals received a 40 mg/kg body weight, subcutaneous, single dose of BOP. Animals in all groups were maintained on a regular protein-containing diet postoperatively, and were then sacrificed 42 weeks after BOP injection. The results showed that a) the incidence of carcinoma in groups 2, 3, and 4 was significantly lower than that in group 1; b) there was no significant difference in group 2 between donor and host pancreas; c) the incidence of carcinoma in the donor pancreas was lower than that in the host pancreas in groups 3 and 4. These results confirm that the protein-free diet significantly inhibits the development of BOP induced pancreatic carcinoma in the hamster. The inhibitory effect appears to act both directly on the pancreas and through an indirect systemic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-oxidized N-nitrosoalkylcarbamates as models for DNA alkylation by N-nitrosobis(2-oxopropyl)amine in Syrian hamsters

A single dose of N-nitrosobis(2-oxopropyl)amine (NDOPA) can selectively induce pancreatic-duct adenocarcinomas in Syrian hamsters. Multiple doses or a higher single dose can induce tumours of the liver and other organs. Our earlier studies employing NDOPA systematically labelled with 14C in the three-carbon chain showed that hamster pancreatic DNA is almost exclusively methylated and that the sole source of the methyl group is the alpha carbon of NDOPA. Hamster liver DNA was equally methylated and alkylated by a three-carbon chain. Current studies using generally labelled tritiated NDOPA with a very high specific activity have shown that the three-carbon alkylation is 2-hydroxypropylation. We have identified two adducts isolated from hamster liver DNA, N7-(2-hydroxypropyl)-guanine and O6-(2-hydroxypropyl)guanine, which contain this group, and we have also isolated and identified N7-methylguanine and O6-methylguanine in DNA from hamster liver and pancreas. beta-Oxidized N-nitrosocarbamates, ethyl N-nitroso-2-oxopropylcarbamate (NOPC) and ethyl N-nitroso-2-hydroxypropylcarbamate (NHPC), are useful models for predicting the DNA adducts observed in vivo following NDOPA treatment. Base-catalysed decomposition of NOPC in the presence of exogenous DNA yields five methylated purines (N3-, N7- and O6-methylguanines and N1- and N3-methyladenines). NHPC, a model for N-nitrosamines containing the 2-hydroxypropyl group, reacts with guanosine to yield N7- and O6-(2-hydroxypropyl)guanines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostatic cancer induced in MRC rats by N-nitrosobis(2-oxopropyl)-amine and N-nitrosobis(2-hydroxypropyl)amine

Weekly intragastric treatment with N-nitrosobis(2-oxo-propyl)amineor N-nitrosobis(2-hydroxypropyl)amine induced hyperplastic,preneoplastic and neoplastic prostatic changes in >80% ofMRC rats. The lesions initially appeared as focal or multifocalproliferations of alveolar epithelium in a cribriform patternwhich, in all but one case, underwent progressive changes, oftentending toward squamous cell formation. Tumors, found primarilyin the ventral prostate, demonstrated various degrees of differentiationand invasive growth. A few neoplasms developed in the seminalvesicles; however all were of a glandular type. The sequentialalteration of induced lesions is described and the possiblereasons for the squamous cell character of most tumors discussed.Prostatic cancer induction by systemic application of specificnitrosamines could provide a unique tool for investigating importantaspects of the disease.     

Induction of pancreatic DNA damage and nodules in rats treated with N-nitrosobis(2-oxopropyl)amine and N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine

DNA damage was demonstrated by alkaline elution analysis inpancreas of rats treated with 100 mg/kg N-nitrosobis(2-oxopropyl)amine(BOP) and 20 mg/kg N-nitroso(2-hydroxy-propyl)2-oxopropyl)amine(HPOP). Young rats were treated with a single dose of BOP orHPOP and autopsied 4 months later. Histologically the pancreasescontained multiple foci and nodules of atypical acinar cellswhich were not seen in controls.     

Subcellular localization of blood group A substance produced by pancreatic adenocarcinoma induced in hamsters by N-nitrosobis(2-oxopropyl)amine (BOP) and by its cell line (PC-1).

The subcellular localization and biochemical characteristics of blood group A antigen were studied by immunogold methods and by SDS-PAGE and Western blotting procedures in N-nitrosobis)2-oxopropyl)amine (BOP)-induced pancreatic cancer (PC) in Syrian hamsters, in the pancreatic cancer cell line (PC-1) derived from a primary induced pancreatic cancer, and in intrapancreatic and subcutaneous transplants of PC-1 cells. Normal hamster duodenal epithelial cells expressing A antigen were compared with the normal hamster pancreas (lacking A antigen), human PC tissues from patients with blood group A and human PC cell lines. Blood group A antigen was present on the membrane of hamster duodenal cells, but was absent in the normal pancreatic cells. A antigen was localized mainly on the cell membrane of the hamster cancer cells both in vivo and in vitro. Glycoproteins with blood group A specificity were observed by SDS-PAGE and Western blotting procedures in the membrane fraction of PC-1 cells, with a major component of molecular mass of approximately 120 kd. Similar migration patterns were observed in the primary induced PC and in subcutaneous and intrapancreatic transplants of PC-1 cells. Membrane preparations from cell lines derived from two primary pancreatic cancers from patients of blood group A and from human pancreatic cell lines, CD11 and CD18, showed a major A reactive component with a molecular mass similar to that found in the hamster PC cells. These findings suggest that: (i) both the hamster and human PC cells in vitro produce glycoproteins with blood group A specificity of similar molecular masses; (ii) differences exist in the structure of the glycoprotein immunoreactive with the anti-A antigen between the normal and cancerous cells; and (iii) differences exist in the molecular mass of the anti-A reactive substance between hamsters and human PC cells and between tissues in vivo and in vitro.     

Effects of 3-aminobenzamide on the post-initiation phase of N-nitrosobis(2-oxopropyl)amine induced pancreatic carcinogenesis in Syrian hamsters.

Effects of 3-aminobenzamide (ABA) on pancreatic carcinogenesis after initiation by N-nitrosobis(2-oxopropyl)amine (BOP) were investigated in Syrian hamsters. Animals were given BOP at a dose of 70 mg/kg body weight by subcutaneous injection and following a 2-week recovery period, were administered basal diet or basal diet containing 0.5, 0.75 and 1.5% ABA for 30 weeks. While the incidences of resultant pancreatic lesions, including hyperplasia, atypical hyperplasia and carcinoma, induced by BOP were not significantly influenced by ABA treatment, the mean numbers of those pancreatic lesions were significantly decreased in a dose-dependent way. The results therefore suggested the possible involvement of poly(ADP-ribosyl)ation in the post-initiation phase of pancreatic carcinogenesis in hamsters.