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Development of hepatocellular carcinomas in rats caused by a choline-deficient, L-amino acid-defined (CDAA) diet, usually associated with fatty liver, fibrosis, cirrhosis and oxidative DNA damage, has been recognized as a useful model of hepatocarcinogenesis caused by endogenous factors. In the present study, in order to further explore involved factors and genes, we established an equivalent model in spontaneous liver tumor-resistant C57BL/6J mice. Six-week-old males and females were continuously fed the CDAA diet and histological liver lesions and oxidative DNA damage due to 8-hydroxydeoxyguanosine (8-OHdG) were examined after 22, 65 and 84 weeks. In male mice, fatty change and fibrosis were evident at 22 weeks, and preneoplastic foci of altered hepatocytes were seen at an incidence of 8/8 (100%) and a multiplicity of 6.6 +/- 4.0 per mouse at 65 weeks. Hepatocellular adenomas and carcinomas developed at incidences of 16/24 (66.7%) and 5/24 (20.8%), and multiplicities of 1.42 +/- 1.32 and 0.29 +/- 0.62, respectively, at 84 weeks. The female mice exhibited resistance to development of these lesions. The CDAA diet also increased 8-OHdG levels in male but not female mice. These results indicate that a CDAA diet causes hepatocellular preneoplastic foci, adenomas and carcinomas associated with fibrosis and oxidative DNA damage in mice, as in rats, providing a hepatocarcinogenesis model caused by endogenous factors in mice.

To elucidate involvement of the transforming growth factor-beta (TGF-beta) signaling pathway in endogenous and exogenous liver carcinogenesis, we investigated mutations of TGF-beta receptor type II (TGF-betaRII), Smad2 and Smad4 genes, and expression of TGF-betaRII in hepatocellular carcinomas (HCCs) induced by a choline-deficient L-amino acid-defined (CDAA) diet and by N-nitrosodiethylamine (DEN). Male Fischer 344 rats received a CDAA diet continuously and HCCs were sampled after 75 weeks. Administration of DEN was followed by partial hepatectomy (PH), with colchicine to induce cell cycle disturbance and a selection pressure regimen, HCCs being obtained after 42 weeks. Total RNAs were extracted from individual HCCs and mutations in TGF-betaRII, Smad2 and Smad4 were investigated by reverse transcription (RT)-polymerase chain reaction (PCR)-restriction-single-strand conformation polymorphism (SSCP) analysis followed by sequencing analysis. Mutations of Smad2 were detected in 2 out of 12 HCCs (16.7%) induced by the CDAA diet, a GGT-to-GGC transition (Gly to Gly) at codon 30 and a TCT-to-GCT (Ser to Ala) transversion at codon 118, without any TGF-betaRII or Smad4 alterations. No mutations of TGF-betaRII, Smad2 and Smad4 were encountered in eleven HCCs induced by the exogenous carcinogen. Semi-quantitative RT-PCR revealed reduced expression of TGF-betaRII in 2 HCCs (16.7%) without Smad2 mutations out of 12 HCCs induced by the CDAA diet and none of 11 induced by DEN. These results suggest that the TGF-beta signaling pathway may be disturbed in endogenous liver carcinogenesis in rats.

Mutations and expression of the transforming growth factor-beta receptor type II (TGF-beta RII) gene were investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Males of the Wistar strain, 6 weeks old, were given 2000 ppm of BHP in their drinking water for 12 weeks and then maintained without further treatment until killed at week 25. Total RNA was extracted from 12 adenocarcinomas and mutations in TGF-beta RII were investigated by RT-PCR-restriction-SSCP analysis followed by sequencing analysis. Two out of 12 adenocarcinomas showed band shifts, indicative of mutations (16.7%). One was a CTG-to-TTG (Leu to Leu) transition at codon 308 without amino acid alteration and the other a frameshift deletion of one of two guanines at nucleotides 1434 to 1435 (codon 477 to 478). Semi-quantitative RT-PCR analysis demonstrated significantly reduced TGF-beta RII expression in adenocarcinomas, as compared with normal lung tissue. These results suggest that TGF-beta RII alterations may play a role in the acquisition of growth advantage by lung adenocarcinomas induced by BHP in rats.

The expression of interleukins (ILs) in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats was investigated using a multiprobe RNase protection assay (RPA) followed by densitometric quantification. Male Wistar rats, 6 weeks old, were given 2000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until they were killed at week 25. Total RNAs were extracted from 14 individual adenocarcinomas and 2 specimens of normal lung tissue of untreated rats. In adenocarcinomas, elevated expression of IL-1alpha (6 / 14), IL-1beta (14 / 14), IL-3 (7 / 14), IL-4 (11 / 14), IL-5 (9 / 14), IL-6 (11 / 14) and IL-10 (8 / 14) was observed, compared with normal lung tissues. In contrast, no expression of IL-2 was detected in any case. The results suggest that preferential expression of these ILs and their complex networks may contribute to the development and progression of lung adenocarcinomas induced by BHP in rats.

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.

We have investigated aberrant methylation of CpG nucleotides (CpG sites) and gene expression of c-myc during hepatocarcinogenesis induced by a choline-deficient, L-amino acid-defined (CDAA) diet in rats. Male Fischer 344 rats, 6 weeks old, were continuously given a CDAA diet for 50 and 75 weeks and then killed. Macroscopically detectable nodules, which were histologically confirmed to be hyperplastic nodules (HNs) or well-differentiated hepatocellular carcinomas (HCCs), were dissected free from the surrounding tissue. Normal control liver was obtained from 6-week-old rats. Methylation of CpG sites of the c-myc gene was investigated in bisulfite-treated DNA isolated from normal liver, HNs and HCCs. All 33 cytosines in the 5'-upstream region of the c-myc gene were fully methylated in control liver and the 4 HNs. In contrast, these cytosines were completely unmethylated in 5 HCCs. Examination of the c-myc expression by reverse transcription-polymerase chain reaction (RT-PCR) analysis also showed a marked increase as compared to the low levels in normal livers and HNs. These results suggest that hypomethylation of the c-myc gene might play a critical role in malignant transformation from HN to HCC during CDAA diet-induced hepatocarcinogenesis in rats.

In the present experiment, we examined the effects of OPB-3206, 3S-[4-(N-hydroxyamino)-2R-isobutylsuccinyl]amino-1-methoxy-3,4- dihydrocarbostyril, a novel metalloproteinase inhibitor, on the growth and metastasis of transplantable osteosarcomas (spontaneous osteosarcoma, selected lung metastatic lesions; S-SLM), which were previously established in rats. OPB-3206 inhibited the activities of interstitial collagenase, gelatinases A and B, and stromelysin in vitro. After oral administration to rats, its serum concentration peaked at 40 min and the drug was no longer detectable at 8 h. When OPB-3206 was orally administered at 0%, 0.1% and 0.4% in the diet for 4 weeks, starting 7 days after subcutaneous transplantation of osteosarcomas to male Fischer 344 rats, numbers of lung metastatic nodules were significantly reduced by the highest dose, while the growth of subcutaneous tumors was not affected. Zymographic analysis showed the presence of pro matrix metalloproteinase (proMMP)-2, proMMP-9 and MMP-9 activities in S-SLM. In animals fed 0.4% OPB-3206, the activity of proMMP-9 was increased, but that for MMP-9 had become undetectable. The results thus suggest that OPB-3206 selectively inhibits lung metastasis of rat transplantable osteosarcomas by inhibiting MMP-9 activation.

We and others have demonstrated that a mutation in Tsc2 is the rate-limiting step for renal carcinogenesis in the Eker rat model. Although inactivation of Tsc2 results in development of renal tumors, it is not sufficient for metastatic renal cell carcinomas (RCs) in the Eker rat. To investigate the additional genetic event(s) necessary for cancer metastasis, we have established highly metastatic S-Lk9d-SLM cell lines from a non-metastatic RC cell line (Lk9dL) by co-implantation with a foreign body (gelatin sponge). Since these cell lines were remarkably different in metastatic performance (all and none, respectively) despite having the same genetic background, they should be useful experimental tools to investigate metastasis-promoting events in renal carcinogenesis.

To determine the role of telomerase activity in the growth of tumors in rats undergoing chemotherapy, a comparison of the volumes of telomerase-positive transplantable osteosarcomas was made in rats treated with the antineoplastic agent cis-diammine dichloroplatinum (CDDP) or the angiogenesis inhibitor O-(chloroacetylcarbamoyl)fumagillol (AGM-1470). Male F344 rats, 8 weeks old, received transplants of macroscopic lung metastatic nodules into the subcutaneous back space and treatment was started on day 14 thereafter. CDDP was injected i.v. at doses of 0, 0.625, 1.25 and 2.5 mg/kg body weight (b.w.) and AGM-1470 was administered at total doses of 0, 2.5, 5 and 10 mg/kg b.w. over 2 weeks by osmotic pumps, also implanted into the subcutaneous back space, but remote from the transplanted tumors. On day 28, all animals were killed for measurement of transplanted tumor size and determination of telomerase activities by telomeric repeat amplification protocol (TRAP) assay. The results showed telomerase activity to be highly correlated with the treated/non-treated (T/C) tumor size ratio (r = 0.96, P < 0.0001). In a second experiment, CDDP at 2.5 mg/kg b.w. and AGM-1470 at 10 mg/kg b.w., these being the most effective doses, were given as in the first experiment, and animals were serially killed on days 14, 21, 28, 35 and 42. Tumors in rats treated with CDDP and AGM-1470 showed 18.2% and 20.5% of the control telomerase activity on days 35 and 21, respectively, when tumor growth was inhibited. However, on day 42, the activities increased to 46.5% and 92.5%, this correlating with re-growth (r = 0.73, P < 0.0001). These results suggest that decline of telomerase activity may be involved in tumor growth retardation induced by chemotherapeutic agents. This possibility clearly warrants further mechanistic studies.

The effects of tomato juice on urinary bladder carcinogenesis were studied in male Fischer 344 rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in rats. The animals (6 weeks old) were given 0.05% BBN in their drinking water for 8 weeks, followed by diluted tomato juice for 12 weeks, and killed at 20 weeks after the beginning of the experiment. Lycopene concentrations in the livers of rats given tomato juice were elevated. Histopathological analysis of urinary bladder lesions revealed the numbers, but not incidences, of urinary bladder transitional cell carcinomas (TCCs) to be decreased in the group given tomato juice. No influence on the incidence of simple and nodullopapillary hyperplasias, invasion or differentiation of TCC was noted. These results indicate that tomato juice, presumably the contained lycopene and other anti-oxidants in combination, exerts an inhibitory effect on the development of TCCs in the rat urinary bladder.

Telomerase is a key enzyme with regard to immortalization of cancer cells and increased activity has been demonstrated in various human malignant neoplasms. Since little is known of its role in pancreatic cancers, we investigated changes in telomerase activity in human pancreatic duct adenocarcinomas and compared the frequency of increased telomerase activity with the presence of K-ras gene mutations. The samples were obtained from 38 pancreatic duct adenocarcinomas and 7 tumor surrounding tissues at surgical resection. Telomerase activity was examined by telomeric repeat amplification protocol assay and terminal restriction fragment (TRF) length was examined by Southern analysis. K-ras mutation was examined by means of polymerase chain reaction-single strand conformation polymorphism analysis. Among 38 pancreatic carcinomas, 32 (84%) exhibited increased telomerase activities with no apparent relation to the histological type of tumor, tumor size, regional lymphnode involvement and distant metastasis or clinical stage. In tissue surrounding the tumor, telomerase activity was not detected. TRF length tended to be reduced in pancreatic carcinomas. Mutations of K-ras gene were found in 24 out of the 38 (63%) cases. Among the 38 cases, 14 showed increased telomerase activity without K-ras mutation and 4 cases showed K-ras mutation without telomerase activity. These results suggest that increased telomerase activity might be a sensitive genetic diagnostic marker and could be a target for future therapy of pancreatic duct carcinomas.

The effects of the non-steroidal anti-inflammatory drug (NSAID) piroxicam and the carotenoids lycopene and beta-carotene, alone or in combination, on the development of rat superficial urinary bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were studied. Male Fischer 344 rats, 6 weeks old, were given 0.05% BBN in the drinking water for 8 weeks followed by administration of piroxicam (0.0075% in the diet), lycopene (0.0025% in the drinking water) and/or beta-carotene (0.0025% in the drinking water) for 12 weeks, then killed for histological analysis of urinary bladder lesions. Cell proliferation potential was analyzed by immunohistochemical staining of the proliferative cell nuclear antigen (PCNA). Piroxicam alone, piroxicam+lycopene, and piroxicam +lycopene+ beta-carotene all significantly decreased the incidences and numbers of transitional cell carcinomas (TCCs), but the combination of piroxicam with carotenoids did not result in a clear improvement in the preventive potential of piroxicam. Piroxicam+ beta-carotene also caused a significant reduction and lycopene alone a slight but not significant reduction in the number of TCCs. In contrast, beta-carotene alone and lycopene+ beta-carotene were without inhibitory influence on any of the lesion categories examined, and the latter significantly increased the proportion of high-grade TCCs. Nevertheless, all of the chemopreventive agents, either alone or in combination, significantly decreased the TCC PCNA index, the effect extending to the surrounding epithelium in the piroxicam+lycopene and piroxicam+lycopene+beta-carotene groups. These results indicate that the NSAID piroxicam may be a more effective chemopreventive agent than lycopene and beta-carotene for superficial urinary bladder carcinogenesis.

Activation of telomerase has been reported in several human cancers, including hepatocellular carcinomas (HCCs). We investigated telomerase activity during hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined (CDAA) diet in rats. Male F344 rats were given a CDAA diet or a choline-supplemented L-amino acid-defined (CSAA) diet from 6 weeks of age for 75 weeks, and subgroups were killed 10 weeks, 50 weeks and 75 weeks after the beginning of the experiment. Hyperplastic nodules and HCCs were noted in rats fed a CDAA diet for 50 weeks and 75 weeks, respectively. Normal control liver specimens were obtained from 6-week-old rats. Telomerase activity was assessed by using a telomeric repeat amplification protocol (TRAP). Normal liver and background parenchyma of rats fed either of the diets for 10 weeks or 50 weeks showed weak telomerase activity. In contrast, markedly increased levels were demonstrated in hyperplastic nodules and HCCs. These results suggest that increased telomerase activity may be a biological feature of preneoplastic lesions that evolve to HCCs in rat liver.

The telomere length in 20 surgically resected human hepatocellular carcinomas (HCCs) and adjacent non-cancerous livers with hepatitis virus infection were investigated. All the HCC samples examined demonstrated shorter telomere length than the corresponding non-cancerous liver tissues, the respective average values being 5.4 kbp and 8.8 kbp (P < 0.001). The shortening of telomere length was most prominent in HCCs larger than 30 mm in diameter, and in both tumors and non-cancerous livers it was more marked with hepatitis B virus as compared with hepatitis C virus infection. These results indicate that telomere shortening is associated with not only progression, but also development of HCC, and there is a possible difference in the nature of the association in patients with hepatitis viruses of B and C types.

The effect of cell cycle disturbance due to colchicine on the induction of enzyme-altered foci during liver regeneration in rats was studied. For initiation, diethylnitrosamine (DEN) at a dose of 10 mg/kg was injected intraperitoneally and partial hepatectomy (PH) was performed 4 h thereafter. Colchicine at doses of 0, 0.1, 0.25 and 0.5 mg/kg was injected intraperitoneally 1 and 3 days after the initiation, followed by application of selection pressure consisting of 2-acetylaminofluorene (AAF) and carbon tetrachloride (CCl4) administration. As end point lesions, gamma-glutamyltransferase (GGT)-positive enzyme-altered foci were assayed at week 5. There was no significant effect of colchicine on numbers of foci. However, a significant, dose-dependent increase in the area of GGT-positive lesions in the groups treated with colchicine was observed. Bromodeoxyuridine labeling indices were higher in foci induced in colchicine-treated rats than in the untreated rats. In a separate experiment, serum glutamic pyruvic transaminase was not increased significantly after DEN and colchicine treatment, and the mitotic index at 6 days after PH was increased in the liver of colchicine-treated rats. These results suggest that the cell cycle disturbance induced by colchicine causes more pronounced selective growth of cells initiated by DEN and colchicine, and this experimental model may be useful for analyzing the mechanisms underlying that growth advantage and the effects of cell cycle abnormalities in liver carcinogenesis.

The effects of methionine on hepatocarcinogenesis induced by coadministration of a choline-deficient L-amino acid-defined (CDAA) diet and ethionine were examined. F344 male rats were divided into 4 experimental groups. Groups 1 and 2 received the CDAA diet and a choline-supplemented L-amino acid-defined (CSAA)++ diet, respectively. Group 3 received the CDAA diet containing 0.05% ethionine, and group 4 the CDAA diet containing 0.05% ethionine and 0.47% methionine. Animals were killed after 12 weeks of treatment. Histologically, the CDAA diet induced intracellular fat accumulation and foci. In contrast, ethionine caused not only foci, but also hyperplastic nodules, cholangiofibrosis and the proliferation of oval cells without such fat accumulation. Methionine abolished the development of all of the liver lesions induced by coadministration of the CDAA diet and ethionine. To investigate the effects of methionine on induction of c-myc and c-Ha-ras expression, as well as generation of 8-hydroxyguanine (8-OHGua) and 2-thiobarbituric acid-reacting substances (TBARS), by coadministration of the CDAA diet and ethionine, subgroups of 3 to 5 animals were killed at 2, 4, 8, or 11 days after the beginning of the experiment. Coadministration of the CDAA diet and ethionine markedly enhanced the level of expression of c-myc and c-Ha-ras, 8-OHGua formation and TBARS generation as compared with the CDAA or CSAA diet within 11 days, and methionine blocks these actions. These results indicate that addition of methionine prevents the induction of c-myc and c-Ha-ras expression, 8-OHGua formation and TBARS generation, as well as hepatocellular lesions, by coadministration of the CDAA diet and ethionine in rats, and suggest a possible involvement of oxidative stress and gene expression in hepatocarcinogenesis by these agents.

The expression of cripto gene product was examined immunohistochemically in 45 surgically resected pancreatic tumors, including 32 invasive ductal carcinomas, 4 intraductal papillary adenocarcinomas, 4 intraductal papillary adenomas, 2 mucinous cystadenomas, 2 islet cell tumors, and one solid and cystic tumor, and compared with that in 32 areas of accompanying chronic pancreatitis present in the cases of invasive ductal carcinomas and 5 non-tumorous areas of pancreas without pancreatitis. All pancreatic ductal tumors including adenomas and carcinomas showed positive staining with no difference in terms of staining intensity among intraductal tumors and invasive carcinomas with or without mucin hypersecretion. Islet cell tumors were positively stained but the solid and cystic tumor was negative. Duct epithelial cells and acinar cells were negative but islet cells were positive in the pancreas tissues without pancreatitis. Cells arranged in duct-like structures in areas of accompanying chronic pancreatitis were positively stained. The results suggest that cripto expression might be associated with a growth advantage of tumor cells and also with differentiation to form duct-like structures.

The presence of K-ras gene mutation was examined in experimentally induced preneoplastic pancreatic ductal lesions. Syrian hamsters received 70 mg/kg of N-nitrosobis(2-oxopropyl)amine (BOP) followed by repeated exposure to an augmentation pressure regimen consisting of choline-deficient diet combined with DL-ethionine and L-methionine and administration of 20 mg/kg BOP. After two augmentation pressure cycles, pancreatic ductal cell hyperplasias appeared and after three cycles, atypical hyperplasias of pancreatic ductal cells and intraductal carcinomas developed. K-ras mutations were detected by single-strand conformation polymorphism analysis of polymerase chain reaction products and nucleotide sequencing. The results showed that K-ras mutation had occurred in one of 9 simple hyperplasias of pancreatic ductal epithelium, in 5 of 9 atypical hyperplasias, and in 4 of 8 intraductal carcinomas. The findings thus suggested that K-ras is activated in association with very early stage malignant transformation of pancreatic ductal cells in hamsters.

The presence of K-ras point mutations in pancreatic duct carcinomas and cholangiocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in Syrian hamsters was investigated by single-strand conformation polymorphism analysis of polymerase chain reaction products from frozen fresh materials in order to clarify the K-ras mutation rates in those two carcinomas induced simultaneously by one carcinogen, BHP. In the examined pancreatic duct carcinomas, 10 out of 16 were positive for a mutation in codon 12 while 3 out of 12 cholangiocarcinomas demonstrated mutation of K-ras gene. G-to-A transition was detected in the second position of codon 12 in both pancreatic carcinomas and cholangiocarcinomas. These results suggest that the role of genetic alteration in carcinogenesis may differ with the target organ, even when initiation is with the same carcinogen.

Effects of inhibitors of arachidonic acid (AA) metabolism on the evolution of preneoplastic foci into nodules and of nodules into hepatocellular carcinomas were examined in F344 male rat livers with or without phenobarbital (PB) exposure. p-Bromophenacyl bromide (BPB), acetylsalicylic acid (ASA), and quercetin (QU) were used as inhibitors of phospholipase A2, cyclooxygenase and lipoxygenase, respectively. Preneoplastic liver foci were induced by initiation with N-nitrosodiethylamine (200 mg/kg, i.p.) followed by selection using the procedure of Cayama et al. For the nodule experiment, starting 1 week after completion of the selection procedure, animals bearing foci were given diets containing 0.05% PB plus 0.75, 1, or 1.5% of one of the inhibitors, 0.05% PB alone, or 0.75, 1 or 1.5% of inhibitor alone, or basal diet for 9 weeks. For the carcinoma experiment, 3 weeks after completion of the selection procedure, animals bearing nodules were given the same diets mentioned above for 29 weeks. BPB, ASA and QU either with or without PB accelerated the remodeling of preneoplastic foci, significantly decreasing the numbers of persistent nodules and hyperplastic nodules. ASA either with or without PB significantly decreased the number of hepatocellular carcinomas per rat. BPB and QU, however, significantly decreased the numbers of hepatocellular carcinomas with but not without PB. The results suggested an involvement of AA metabolism in the process of evolution of preneoplastic foci into nodules and hepatocellular carcinomas in rat liver with or without PB exposure.

Eighty cases of cystectomized and step-sectioned pT1 transitional cell carcinomas of various grades were analyzed. During the same period, 30 consecutive cases of pT2 carcinomas were also cystectomized and examined for comparison. This is a cross-sectional study of a variety of cases of bladder cancer, designed to delineate the characteristics of G3.pT1 disease. Tumors in this series had a full set of various pathological findings; grades 1 to 3, stages Ta-T1-T2, papillary-papillonodular-nodular tumor configuration, alpha-beta-gamma type of invasion, presence of lymphatic and venous involvement, and presence of associated carcinoma in situ/dysplasia. There is a distinct tendency of stepwise disease progression, such as grade 1-->3, stage T1-->T2, papillary-->nodular configuration, and alpha-->gamma invasion, these factors being mutually related. We noted a similarity between tumor groups containing G3 component, such as G2-3.pT1, G3 > 2.pT1, G3.pT1, G1-3.pT2, and G3.pT2. These tumors are different from groups such as G1-2.pT1, G2.pT1 and G2 > 3.pT1 in terms of tumor configuration and type of invasion. As regards pT1 having G3 components, early cystectomy seems to be the surest treatment. Whenever one adopts a conservative policy in treatment of these tumors, extreme care should be taken to monitor tumor progression.

The possibility of poly(ADP-ribosyl)ation playing a role during liver regeneration induced by partial hepatectomy (PH) in vivo was examined. When rats were given an i.p. injection of 3-aminobenzamide (ABA) at a dose of 600 mg/kg body weight 12 h after PH, the levels of DNA synthesis at 20 h after PH were significantly reduced. The time course of DNA synthesis in regenerating liver was significantly delayed in the ABA-treated group. Enzymatic assay revealed the activity of poly-(ADP-ribose)polymerase (PADPRP) in controls to be increased in parallel with the increase of DNA synthesis induced by PH. This increase in PADPRP activity was delayed and very much weaker after ABA treatment. The results thus suggested that poly(ADP-ribosyl)ation might play an important role in DNA synthesis during liver regeneration in vivo.

Five pancreatic carcinomas induced by N-nitrosobis(2-hydroxypropyl)amine in Syrian golden hamsters were analyzed for activation of Ki-ras at codons 12 and 13, using the polymerase chain reaction and direct sequencing. The Ki-ras gene was shown to be activated in four of the five carcinomas, and the results were further confirmed by subcloning and sequencing. All the mutations involved a G-to-A transition at the second position of codon 12, which resulted in a change at the amino acid level from glycine to aspartic acid. This mutation is identical with that reported for pancreatic tumors of Syrian hamsters induced by N-nitrosobis(2-oxopropyl)amine.

The effects of an inhibitor of poly(ADP-ribose)polymerase, 3-aminobenzamide (ABA), on N-nitrosobis(2-hydroxypropyl)amine (BHP)-induced pancreas, liver, gallbladder and lung carcinogenesis in Syrian golden hamsters were investigated. Animals were given either BHP alone, by subcutaneous injection at a dose of 500 mg/kg body weight, or in combination with an intraperitoneal injection of ABA 30 min after the BHP at a dose of 300 or 600 mg/kg body weight once a week for 5 weeks, and then killed 35 weeks after the commencement of the experiment. ABA exerted inhibitory effects on pancreas and lung carcinogenesis induced by BHP, with mean numbers of lesions (including hyperplasias and carcinomas) being significantly decreased compared with the BHP-alone group values, while no significant effect was observed on liver or gallbladder carcinogenesis. These results suggest that the effects of ABA on carcinogenesis depend on the target organ as well as the chemical carcinogen examined.

The potential initiating activity of diethylnitrosamine (DEN) was studied in a rapid production model for pancreatic carcinogenesis in hamsters developed in our laboratory incorporating the principle of selection based on resistance of cytotoxicity, originally demonstrated for liver carcinogenesis in rats. Female Syrian golden hamsters were given DEN at a dose of 100 mg/kg body weight or N-nitrosobis-(2-oxopropyl)amine (BOP) at a dose of 70 mg/kg body weight as initiators followed by 3 cycles of augmentation pressure (choline-deficient diet combined with DL-ethionine, L-methionine upon return to basal diet and then administration of 20 mg/kg body weight BOP), and killed 10 weeks after the beginning of the experiment. DEN followed by the augmentation pressure induced a 65% incidence of total pancreatic lesions including 15% carcinomas, while BOP followed by the augmentation pressure induced 100% incidence of total pancreatic lesions and 84.2% for carcinomas. These yields were significantly greater than those observed for augmentation pressure alone. The results thus indicate that DEN possesses weak initiating activity for pancreatic carcinogenesis under the present experimental conditions.

The content of glutathione S-transferase placental form (GST-pi) was immunohistochemically analyzed in 100 cases of human stomach carcinoma of various histological types and compared with the content in the stomach mucosa of the human fetus, infant and adult. High levels of GST-pi content were demonstrated in all stomach carcinomas except for signet ring cell carcinomas. High levels were also present in surface mucous cells and glandular cells of the stomach from a fetus aged 18 weeks, whereas only the parietal cells of fundic glands in the adult stomach showed slight staining for GST-pi. These results indicate that the phenotypic expression of GST-pi in human stomach carcinoma is oncofetal in character.

The hst-1 gene, previously designated the hst gene, was identified as a transforming gene by transfection assays of genomic DNAs from various types of human cancers. We analyzed for alterations of the hst-1 gene in 18 bladder cancers, 23 renal cell carcinomas and 5 esophageal cancers. Although rearrangement of the gene was not detected in any of these samples, amplification of the hst-1 gene was found in 4 samples of tumors, including an invasive bladder cancer, both primary esophageal cancer and its lymph node metastasis, and kidney metastasis of an esophageal cancer. The same degree of amplification of the int-2 gene, the product of which has significant homology with the hst-1-encoded protein, was also observed in all of these DNA samples with amplified hst-1 gene. This result indicates close chromosomal localization of the two genes, which were amplified as one amplification unit.

Both spontaneous (SOS) and 4-hydroxyaminoquinoline 1-oxide (4-HAQO)-induced osteosarcomas (COS) could be serially transplanted in the subcutaneous back space of syngeneic F344 rats, the success rate becoming 100% within 5 passage generations. Transplanted tumors demonstrated rapid growth and displayed high potential for metastasis to the lung in later generations. Thus, a 100% lung metastasis rate was observed for SOS after the 20th and for COS after the 14th generation. The histological features of the primary SOS and COS were retained during serial transfer. These model systems should be useful for investigation of the biology of this very important tumor type.

Nicotinamide adenine dinucleotide is utilized as the substrate of a chromatin-bound enzyme, poly(ADP-ribose) polymerase. The effects of diethylnitrosamine and/or 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase, on the cellular NAD levels in rat liver were investigated. 3-Aminobenzamide (600 mg/kg) administered intraperitoneally was not detectable in the liver within 12 hr after administration; the inhibitor had a calculated half life of 90 min. Diethylnitrosamine reduced the NAD levels in rat liver in a dose-dependent way. The NAD content reached a minimum level at 8 hr, returning to 78% of the control value after 48 hr. The reduction of the NAD levels caused by diethylnitrosamine was completely prevented when 3-aminobenzamide was administered either simultaneously with diethylnitrosamine or 4 hr after diethylnitrosamine treatment. Furthermore, an immunohistochemical study showed that nuclear poly(ADP-ribose) decreased 1 hr after the administration of 3-aminobenzamide. These results suggest that inhibition of poly(ADP-ribosyl)ation is involved in the initiation of liver carcinogenesis by diethylnitrosamine and 3-aminobenzamide.

The liver metastatic potential of alpha-fetoprotein (AFP)-producing human gastric carcinoma (NSC-3) was examined in male, BALB/c, nude mice. Metastatic nodules in the liver were produced by intrasplenic (IS) injection of tumor cell suspension prepared by trypsinization from subcutaneous NSC-3 tumor. The serum AFP level increased exponentially after IS injection along with the growth of metastatic nodules in the liver, and a positive correlation was observed between the estimated weight of metastatic nodules and serum AFP level. To investigate the effect of liver damage by carbon tetrachloride (CCl4) on the metastatic potential of NSC-3 cells injected intrasplenically, the mice were divided into 4 groups: Group 1 received IS injection of 1 x 10(6) of NSC-3 cells without CCl4 treatment; Groups 2, 3 and 4 received IS injection 7 days, 2 days and 1 day after CCl4 treatment, respectively. All mice were killed 64 days after IS injection. The incidence of liver metastasis was 80% in Group 1, but 100% in Groups 2, 3 and 4. The mean numbers of metastatic nodules per liver were 4.2 in Group 1, 16.8 in Group 2, 18.0 in Group 3 and 44.5 in Group 4. Significant differences in the mean numbers of metastatic nodules were observed between Group 4 and the other groups. It was clearly demonstrated that the metastatic potential of AFP-producing human gastric carcinoma cells (NSC-3) is enhanced in the situation prevailing after liver parenchymal cells are damaged by CCl4.