Detection of the Hepatitis B Virus X Protein (HBx) Antigen and Anti-HBx Antibodies in Cases of Human Hepatocellular Carcinoma

Abstract

Hepatitis B virus (HBV) infection can cause a wide spectrum of sequelae, ranging from asymptomatic chronic infection to chronic active hepatitis. Three hundred million people around the world are chronically infected with HBV (38, 39). Clinically, it is estimated that 5 to 10% of HBV-infected adults will develop chronic hepatitis (CH) and will thus be at a higher (Ͼ100 times) risk of developing hepatocellular carcinoma (HCC) (1,12,41). Several viral and host factors have been suggested to be involved in the chronicity and diversity of HBV-associated disease (25, 43). The association, at the molecular level, between HBV infection and HBV-related diseases remains unclear.The small, 3.2-kb DNA genome of HBV contains four known open reading frames, called S, C, P, and X. The hepatitis B virus X (HBx) gene is the smallest, with a length of 465 nucleotides (10). HBx protein is 154 amino acids long, with a molecular weight (MW) of 17,000, and is conserved among all mammalian hepadnaviruses, but the deduced amino acid sequence of the HBx gene product does not correspond to any known viral proteins (26). Although the HBx protein has been shown to stimulate cell cycle progression of quiescent cells (18), it is mainly a pleiotropic transactivator, due to its ability to stimulate not only the HBV promoter and enhancer (28, 33) but also a wide range of other viral promoters (33,35,40). The necessity for HBx gene expression during the viral life cycle, in vitro and in vivo, has been suggested (3, 44). The HBx protein acts either through interaction with other cellular transcription factors or via a signal transduction pathway controlled by protein kinase C (15, 30, 31). As a consequence of its activity, the HBx protein appears capable of inducing transformation (32) and liver tumors in a selected strain of mice that express the HBx protein from a transgene (16,17).During the natural course of HBV infection, the HBx gene expresses a polypeptide, HBx, that is implicated in HBV-mediated HCC (4, 16). When liver tissue samples from HCC and CH patients were reacted with an anti-HBx antibody and evaluated by immunohistochemistry, reactive antigen was detected in 80% of HCC liver samples and 30% of CH liver samples (4). In another study, the sera of patients with acute hepatitis, CH, and cirrhosis were tested for HBx protein and anti-HBx antibodies by an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody and recombinant HBx protein.The results indicated that 23% of patients' sera were HBx positive and 14% of patients' sera were anti-HBx positive (20). In another approach, using HBx oligopeptides as antigens to detect antibodies in the sera of HCC patients, 73% of HCC sera tested positive for anti-HBx antibodies (27). With similar approaches, data showed that 74% of sera from patients with cirrhosis and 54% of sera from patients with HCC were positive for anti-HBx antibodies and HBV surface antigen (HBs) (36). Therefore, the expression of HBx protein in infected patients did not correlate well with the occurrence of...

Transformation of human liver L-O2 cells mediated by stable HBx transfection

“…It has been reported that HBx is preferentially maintained in HCC in the absence of other viral antigens [25] . However, the function of HBx in the transformation of human liver cells is unknown.…”

“…The hepatocyte cell line L-O2 (Nanjing KeyGen Biotech Co Ltd, Nanjing, China), originating histologically from normal human liver tissue immortalized by stable transfection with the hTERT gene, has been used previously [21,[24][25][26][27] . L-O2-P (a cell line derived from L-O2 cells stably transfected with an empty pcDNA3 plasmid) and L-O2-X (a cell line derived from L-O2 cells stably transfected with the HBx gene) were established as described previously [28] .…”

Hepatitis B virus-related hepatocellular carcinoma: paradigms for viral-related human carcinogenesis

“…HBx is expressed at low levels during acute and chronic hepatitis and induces a humoral and cellular immune response (Chung et al, 1999;Hwang et al, 2002;Chun et al, 2003;Malmassari et al, 2005). In some studies, antiHBx antibodies were more frequent among patients with HCC than those with chronic hepatitis B without cancer (Hess et al, 1988;Levrero et al, 1991;Hwang et al, 2003). In most integrated subviral DNA, the HBx gene is maintained and transcribed and several studies have now demonstrated both HBx RNA and protein expression in human HCC tumor cells in the absence of HBV replication (Su et al, 1998;Peng et al, 2005).…”

Signal transduction cascades and hepatitis B and C related hepatocellular carcinoma

“…Studies have found that patients with HCC produce HBx-specific antibodies with much greater frequency than patients with chronic HBV infection alone, with or without cirrhosis (70% vs. 5-15% respectively) [86][87][88] In addition, HBx RNA and protein can be demonstrated in HCC tissue samples. 81,86,87,89 Animal studies have provided evidence which implicates HBx in hepatocyte transformation and oncogenesis. Transgenic mice expressing HBx established by Koike et al frequently develop liver neoplasms (including HCC), particularly those demonstrating high levels of X gene product.…”