Ccne1 Overexpression Causes Chromosome Instability in Liver Cells and Liver Tumor Development in Mice.

BACKGROUND & AIMS: The CCNE1 locus, which encodes cyclin E1, is amplified in many types of cancer cells and is activated in hepatocellular carcinomas (HCCs) from patients infected with hepatitis B virus or adeno-associated virus type 2, due to integration of the virus nearby. We investigated cell-cycle and oncogenic effects of cyclin E1 overexpression in tissues of mice. METHODS: We generated mice with doxycycline-inducible expression of Ccne1 (Ccne1T mice) and activated overexpression of cyclin E1 from age 3 weeks onward. At 14 months of age, livers were collected from mice that overexpress cyclin E1 and nontransgenic mice (controls) and analyzed for tumor burden and by histology. Mouse embryonic fibroblasts (MEFs) and hepatocytes from Ccne1T and control mice were analyzed to determine the extent to which cyclin E1 overexpression perturbs S-phase entry, DNA replication, and numbers and structures of chromosomes. Tissues from 4-month-old Ccne1T and control mice (at that age were free of tumors) were analyzed for chromosome alterations, to investigate the mechanisms by which cyclin E1 predisposes hepatocytes to transformation. RESULTS: Ccne1T mice developed more hepatocellular adenomas and HCCs than control mice. Tumors developed only in livers of Ccne1T mice, despite high levels of cyclin E1 in other tissues. Ccne1T MEFs had defects that promoted chromosome missegregation and aneuploidy, including incomplete replication of DNA, centrosome amplification, and formation of nonperpendicular mitotic spindles. Whereas Ccne1T mice accumulated near-diploid aneuploid cells in multiple tissues and organs, polyploidization was observed only in hepatocytes, with losses and gains of whole chromosomes, DNA damage, and oxidative stress. CONCLUSIONS: Livers, but not other tissues of mice with inducible overexpression of cyclin E1, develop tumors. More hepatocytes from the cyclin E1-overexpressing mice were polyploid than from control mice, and had losses or gains of whole chromosomes, DNA damage, and oxidative stress; all of these have been observed in human HCC cells. The increased risk of HCC in patients with hepatitis B virus or adeno-associated virus type 2 infection might involve activation of cyclin E1 and its effects on chromosomes and genomes of liver cells.

Use of RNA interference to modulate liver adenoma development in a murine model transgenic for hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is closely related to the development of severe liver complications, including hepatocellular carcinoma. In previous studies, we reported that in vivo long-term HBV suppression in transgenic mice can be achieved without apparent toxicity by short hairpin RNA sequentially delivered using adeno-associated viral (AAV) vectors of different serotypes. Our goal herein was to address the clinical utility of this delivery system and, in particular, to determine whether RNA interference (RNAi) and its ability to induce long-term HBV suppression will modulate the development of HBV-associated liver pathology. As a model system, we used a unique HBV transgenic mouse model, containing a 1.3 times over length of the HBV genome, on the ICR mouse background. These transgenic mice produce high serum HBV titers comparable with human chronic HBV patients, and, importantly, manifest characteristic HBV-associated pathology, including progressive hepatocellular injury and the development of hepatocellular adenoma. Using this system, we injected animals with AAV vectors expressing either HBV-specific or a control luciferase-specific short hairpin RNA and followed animals for a total of 18 months. We report herein that AAV-mediated RNAi therapy profoundly inhibits HBV replication and gene expression, with a significant reduction in hepatic regeneration, liver enzymes and, importantly, the appearance of liver adenomas. Indeed, the therapeutic effect of RNAi correlated with the reduction in HBV titers. Our data demonstrate that appropriately designed RNAi therapy has the potential to prevent formation of HBV-associated hepatocellular adenoma.

Influence of ursodeoxycholate-enriched diet on liver tumor growth in HBV transgenic mice.

Hepatitis B virus (HBV) transgenic mice (official designation, Tg [Alb-1 HBV] Bri 44) invariably develop macroscopically evident tumors within the 20th month of life. Sustained proliferative activity seems to play an important role in the development of these lesions. We previously showed that ursodeoxycholate (UDC) stimulates hepatocyte proliferation in various experimental settings. Herein, we tested the assumption that biological factors able to further increase liver cell proliferation, such as UDC, could accelerate tumor development in this animal model. For this study, 22 eight-week-old male transgenic mice were divided into 2 groups; 11 animals received a standard diet, and 11 received a UDC-enriched diet. The 2 groups were further divided into 2 subgroups of 5 and 6 animals each and were sacrificed at 3 and 15 months of age, respectively. These different times were chosen to exclude diet-related toxicity (in 3-month-old mice) and evaluate tumor growth (in 15-month-old mice). In addition, hepatocyte proliferation was assessed in all animals. In 3-month-old mice receiving UDC, cholestatic and cytolytic indices as well as liver histology were comparable to those in controls. At 15 months, all UDC-treated mice showed large multinodular tumors whereas only 33% of controls developed smaller uninodular neoplasms. Hepatocyte proliferation was increased in all animals receiving UDC compared with controls. In conclusion, the increase in serum UDC (undetectable in mice fed a standard diet), in the absence of any toxic effect on the liver, suggests the involvement of this bile salt in the stimulation of hepatocyte proliferation and tumor growth.

Woodchuck hepatitis virus replication and antigen expression gradually decrease in preneoplastic hepatocellular lineages.

BACKGROUND/AIMS: Hepatocellular carcinomas elicited in woodchucks by the woodchuck hepatitis virus (WHV) emerge gradually from parenchymal areas of minimal structural deviation via two predominant preneoplastic hepatocellular lineages, composed of either glycogenotic/basophilic or amphophilic/basophilic cell foci. In this study we analyzed WHV replication during neoplastic development in both lineages. METHODS: In minimal deviation areas, preneoplastic hepatocellular foci, and hepatocellular neoplasms, developing in 16 WHV-carriers 31-38 months after WHV-inoculation, the proportion of hepatocytes containing WHV replicative intermediates (as detected by in situ hybridization for WHV DNA) and immunoreactive for WHV core and surface antigens was assessed. RESULTS: Appearance of WHV replicative intermediates and expression of antigens were limited to the cytoplasm of hepatocytes and were strongly correlated (P<0.0001), both showing high levels in minimal deviation areas, but markedly reduced amounts in all types of preneoplastic hepatic focus (P<0.0001), and in hepatocellular adenomas. Most hepatocellular carcinomas were negative for WHV replicative intermediates and antigens. CONCLUSIONS: In both the glycogenotic-basophilic and the amphophilic-basophilic preneoplastic hepatocellular lineage, WHV replication and antigen expression gradually decrease early during the preneoplastic phase. The close correlation of these changes with metabolic aberrations characterizing preneoplastic hepatocellular lineages suggests that oncogenic effects mimicking insulin/glucagon imbalances may be responsible for the repression of hepadnaviral replication.