Marcy Clayton

Hepatitis B virus transgenic mouse model of chronic liver disease

A model for hepatitis B virus-associated chronic liver disease has been made using cloned hepatitis B virus DNA as a transgene in a severe combined immunodeficient host. These mice consistently support virus gene expression and replication. After adoptive transfer of unprimed, syngeneic splenocytes, these mice cleared virus from liver and serum, and developed chronic liver disease. This model will permit identification of the host and virus contributions to chronic liver disease in the absence of tolerance.

Enhanced cell survival of Hep3B cells by the hepatitis B x antigen effector, URG11, is associated with upregulation of β‐catenin

Intrahepatic expression of hepatitis B x antigen (HBxAg) is associated with the development of hepatocellular carcinoma (HCC), perhaps through trans‐activation of selected cellular genes. When this was examined by PowerBlot analysis, upregulated levels of β‐catenin and several known β‐catenin effectors were observed in HBxAg‐positive compared with HBxAg‐negative HepG2 cells. When HBxAg was introduced into Hep3B cells, upregulated expression of wild‐type β‐catenin was observed. This was also observed in Hep3B cells overexpressing the HBxAg upregulated gene, URG11. Upregulated expression of URG11 and β‐catenin correlated with HBxAg trans‐activation function. Transient transfection assays with fragments of the β‐catenin promoter showed that it was activated by both HBxAg and URG11 and inhibited by URG11‐specific small inhibitory RNA. The latter also inhibited the growth of Hep3BX cells in a serum‐free medium, which correlated with depressed levels of β‐catenin. Activation of β‐catenin effector genes was observed in cells stably expressing HBxAg or overexpressing URG11 compared with control cells transfected with the pTOPFLASH reporter plasmid. Extensive costaining between HBxAg, URG11, and β‐catenin was observed in infected liver and HCC nodules, suggesting a close relationship in vivo. In conclusion, wild‐type β‐catenin is activated by HBxAg, in part, through the upregulated expression of the HBxAg effector URG11. URG11 stimulates the β‐catenin promoter and hepatocellular growth and survival. These observations also suggest that URG11 may be a regulatory element in the β‐catenin signaling pathway and may be a target for chemoprevention of HCC. (HEPATOLOGY 2006;43:415–424.)

Upregulated Expression of a Unique Gene by Hepatitis B x Antigen Promotes Hepatocellular Growth and Tumorigenesis

Hepatitis B x antigen (HB x Ag) is a trans-activating protein that may be involved in hepatocarcinogenesis, although few natural effectors of HB x Ag that participate in this process have been identified. To identify additional effectors, whole cell RNA isolated from HB x Ag-positive and HB x Ag-negative HepG2 cells were compared by polymerase chain reaction select cDNA subtraction, and one clone, upregulated gene, clone 11 (URG11), was chosen for further characterization. Elevated levels of URG11 mRNA and protein were observed in HB x Ag-positive compared to HB x Ag-negative HepG2 cells. Costaining was observed in infected liver (P < 0.01). URG11 stimulated cell growth in culture (P < 0.01), anchorage-independent growth in soft agar (P < 0.001), and accelerated tumor formation (P < 0.01), and yielded larger tumors (P < 0.02) in SCID mice injected subcutaneously with HepG2 cells. These data suggest that URG11 is a natural effector of HB x Ag that may promote the development of hepatocellular carcinoma.

Hepatitis C virus replication in stably transfected HepG2 cells promotes hepatocellular growth and tumorigenesis

HepG2 cells stably transfected with a full‐length, infectious hepatitis C virus (HCV) cDNA demonstrated consistent replication of HCV for more than 3 years. Intracellular minus strand HCV RNA was present. Minus strand synthesis was NS5B dependent, and was sensitive to interferon alpha (IFNα) treatment. NS5B and HCV core protein were detectable. HCV stimulated HepG2 cell growth and survival in culture, in soft agar, and accelerated tumor growth in SCID mice. These mice became HCV RNA positive in blood, where the virus was also sensitive to IFNα. The RNA banded at the density of HCV, and was resistant to RNase prior to extraction. Hence, HCV stably replicates in HepG2 cells, stimulates hepatocellular growth and tumorigenesis, and is susceptible to IFNα both in vitro and in vivo.