Keiko Ohtake

Hepatocarcinogenesis in Transgenic Mice Carrying Albumin‐promoted SV40 T Antigen Gene

We have developed transgenic mice that inherit albumin promoter‐regulated simian virus 40 (SV40) large T antigen gene, expressed specifically in hepatocytes. These mice all develop multifocal hepatocellular carcinomas at around 5 months and die of liver insufficiency by 7 months. Sequential morphological observation of hepatocarcinogenesis revealed 5 distinct stages: (I) newborn to 2 weeks of age, neither recognizable histological changes nor cellular replication in spite of T antigen expression; (II) between 3 and 7 weeks, diffuse cytomegalic change of hepatocytes with numerous abnormal mitoses, usually resulting in cell death; (III) from 7 weeks onwards, quasi‐regenerative small hepatocyte foci with a decreased tendency for cytomegaly in spite of T antigen expression, rapidly replacing the hepatic tissue; (IV) 11 weeks of age and thereafter, neoplastic foci and nodules with enzymatic alteration; (V) 20 weeks of age and thereafter, gross hepatocellular carcinomas with occasional pulmonary metastases. Considerable variation existed both in morphological and enzymatic features and T antigen expression among neoplastic lesions, including carcinomas. Thus, these transgenic mice clearly show a multistep process in hepatocarcinogenesis with remarkable synchrony and provide a promising model for analyzing the essential events of carcinogenesis at different stages.

Instability of integrated hepatitis B virus DNA with inverted repeat structure in a transgenic mouse

We established four cell lines, from the liver cells of a transgenic mouse, constructed with hepatitis B virus DNA that had an inverted repeat structure. The integrated DNA patterns of the four established cell lines were different from one another and from the original pattern. These data show that the instability of integrated hepatitis B virus DNA would also occur in somatic cells during replication, apart from meiosis, which was previously reported.

Absence of H-ras point mutation at codon 12 inN-methyl-N-nitrosourea-induced hepatocellular neoplasms in the rat

SummaryIn order to assess the possibility that activatedras-associated hepatic carcinomas might be much rarer in rats than mice because of the more frequent or rapid occurrence of powerful carcinogenic event(s) other thanras point mutations in the former animals, precancerous lesions and hepatocellular carcinomas induced by a weak hepatocarcinogenN-methyl-N-nitrosourea (MNU) in the rat liver were analyzed for the presence or absence ofras point mutations. MNU was chosen because it is well known that MNU-induced rat mammary carcinomas contain activated H-ras at very high frequency. Male Fisher rats were treated with a single dose of MNU after partial hepatectomy, and then administered dietary phenobarbital or repeated s.c. injections of carbon tetrachloride as promoting procedures. Analyses by oligonucleotide hybridization, MnlI-restriction-fragment-length polymorphism and NIH3T3 cell transfection assays revealed neither H-ras point mutations nor transforming ability of the DNA from 36 MNU-induced rat hepatic neoplasms. The results were in agreement with previous results for rat hepatocellular carcinomas induced by other potent liver carcinogens and did not support our hypothesis that the frequency of findingras activation might be dependent on the strength of the carcinogen.

In vitro Progression‐associated c‐H‐ras Activation in Neoplastic Hepatocyte Lines Established from SV40‐T Antigen Gene‐harboring Transgenic Mice

In order to investigate the molecular mechanisms of multistep hepatocarcinogenesis in SV40‐T antigen gene‐harboring transgenic mice, 9 hepatocellular carcinoma (HCC) lines and 10 “preneoplastic” hepatocyte lines were established from the animals and their biological and molecular changes during culture were investigated. Three of the 9 HCC lines showed progression during culture in terms of growth rate and growth capability in soft agar and in nude mice. This progression was associated with the appearance of activated c‐H‐ras oncogene. Including these 3 lines, H‐ras activation was observed in a total of 7 of the 9 HCC lines (78%), whereas it was found only in 1 of 10 (10%) “preneoplastic” hepatocyte lines. These data thus indicate that H‐ras activation may be an event occurring at a relatively late stage of hepatocarcinogenesis in this transgenic mouse system and that it may serve towards completion of the carcinogenic process together with the T‐antigen.

Frequent spontaneous sister chromatid exchange in hepatocytes of transgenic mice harboring the SV40-T antigen gene

SummaryIn order to shed light on the causal mechanisms of hepatocarcinogenesis in the transgenic mouse into which the albumin-promotor-regulated SV40-T antigen gene has been introduced (T+ mouse), and especially on the frequent chromosomal aberrations seen in cultured hepatocytes and hepatocellular neoplasms derived from such animals, the frequency of sister chromatid exchange (SCE) and karyotype abnormalities were investigated in a hepatocyte primary culture system. Cells were obtained through collagenase perfusion from T+ mice at 16–18 days of age, when no morphological changes are apparent, and from nontransgenic littermates, and cultured in the presence of bromodeoxyuridine. SCE was seen in transgenic hepatocytes twice as frequently as in their normal counterparts. No karyotype abnormalities in terms of numerical change or gross aberration were detected at this phase. The results thus suggest mutagenic properties for the T antigen, which may play an important role in hepatocarcinogenesis in this transgenic mouse.

Subcutaneous sarcomas of probable neuronal origin in a transgenic mouse strain containing an albumin promoter-fused simian virus 40 large T antigen gene

Frequent development of subcutaneous neurogenic sarcomas was observed in a hepatocellular carcinoma‐producing transgenic mouse strain harboring an albumin‐promoted simian virus 40 (SV40) large T antigen gene. Found unexpectedly in 19 out of 306 mice (6.2%) by 6 months of age, all the sarcomas were similar and were characterized as neurogenic on the basis of histological features including Homer‐Wright type rosette formation, the presence of dense core granules of 100–200 nm diameter under the electron microscope, expression of neuron specific enolase, S‐100 protein, and catecholamines, and nerve cell‐like differentiation in culture in response to But2cAMP, Immunohistochemical study revealed tiny clusters of SV40 T antigen‐expressing cells with neurogenic character in normal‐appearing adult mouse subcutis as candidate progenitors of the sarcomas. The tumor cells strongly expressed large T antigen but did not express albumin or albumin mRNA at the detection sensitivity used. Transient transfection assay (CAT assay), however, revealed the presence of transcriptional factor(s) acting on the albumin promoter in tumor cells. Thus, the present investigation suggested the presence of specifically differentiated neurogenic cells in the mouse subcutis with aberrant expression of the transgene.

Development of transgenic mice containing woodchuck hepatitis virus DNA

SummaryWe produced transgenic mice containing woodchuck hepatitis virus DNA with tandem repeat structure capable of producing virus and viral antigens. Poly(A)+ RNAs probably corresponding to pregenome and viral antigens were detected in their liver. These mice have now been healthy for one year. However, there is the possibility of inducing immunologically mediated hepatitis. We anticipate these transgenic mice may present a useful model system for studying the significance of chronic hepatitis in hepatocellular carcinoma development.