Dominance of functional androgen receptor allele with longer CAG repeat in hepatitis B virus-related female hepatocarcinogenesis.

The CAG polymorphism in exon 1 of the androgen receptor (AR) gene has been shown associated with the development of human male hepatocellular carcinoma (HCC) with the shorter AR alleles conferring a higher risk. However, the significance of AR-CAG repeats in female hepatocarcinogenesis remains to be addressed. In this study, seventy-six pairs of female HCCs and corresponding nontumorous tissues were collected, and 180 cirrhotic nodules were microdissected from 7 cirrhotic livers. The clonality status, functional AR alleles, and CAG repeat number of each sample were determined by AR methylation analysis. In a total of 44 monoclonal HCCs, the mean of CAG repeats in the active alleles was significantly longer than that in the inactive alleles (22.0 +/- 2.8 versus 20.7 +/- 3.6; P = 0.047). When we divided HCCs into hepatitis B virus-positive [HBV(+)] and HBV(-) subgroups, the long AR allele dominance was found only in HBV(+) ones (P = 0.006 versus P = 0.923). Notably, the preference of long CAG repeat has also been found in the 100 monoclonal nodules (P = 0.013). For comparison of monoclonal nodules obtained from the same individual, a dominant long AR allele was found in 6 patients. The proportion of monoclonal cirrhotic nodules and HCCs expressing longer AR allele, 69 and 68%, are both significantly higher than 50%, the assumed value in normal liver (P < 0.001 for cirrhotic nodules and P = 0.005 for HCC). The dominance is again only prominent in HBV-infected HCCs [85% for HBV(+) HCC; P < 0.001 but 54% for HBV(-) HCC; P = 0.27]. The results indicated that in female hepatocarcinogenesis, hepatocytes expressing the longer AR allele seem to be favorably selected for autonomous growth and transformation, especially in synergy with HBV infection.

High-throughput cell-based screening for hepatitis C virus NS3/4A protease inhibitors.

Hepatitis C virus (HCV) encodes a viral protease, nonstructural (NS)3/4A, that is critical for virus maturation. Although NS3/4A has emerged as a promising target for anti-HCV drug discovery, no anti-HCV therapy has succeeded yet based on inhibition of NS3/4A. We have previously shown that EG(delta4AB)SEAP, a reporter consisting of enhanced green fluorescent protein (EG), the NS3-NS4A protease decapeptide recognition sequence (delta4AB), and secreted alkaline phosphatase (SEAP), is an efficient reporter for reflecting NS3/4A proteolytic activity inside cells. In this study, we describe the generation and characterization of a stable cell line, 293EEG(delta4AB)SEAP-NS3/4A, which constitutively expresses EG(delta4AB)SEAP reporter protein and NS3/4A protease. The reporter assay is validated with the compound BILN 2061, a specific and potent peptidomimetic inhibitor of the HCV NS3 protease. Additionally, we show here that this cell line allows screening for NS3/4A protease activity of living cells in 96-well plate format, with a Z factor >0.6. Thus, this cell-based assay may be used for high-throughput screening of chemical libraries.

A reporter-based assay for identifying hepatitis C virus inhibitors based on subgenomic replicon cells.

Hepatitis C virus (HCV) encodes a polyprotein that needs to be processed proteolytically by cellular and viral proteases into mature functional proteins. One of the viral proteins, NS3/4A, has serine protease activity that is critical for virus maturation. The generation and characterization of an engineered HCV replicon cell line (Ava5) is described which constitutively expresses EGdelta4AB)SEAP reporter protein and the cell line was designated as Ava5-EG(delta4AB)SEAP. EG(delta4AB)SEAP is a fusion protein in which Enhanced Green Fluorescent Protein (EGFP) was fused to SEcreted Alkaline Phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, delta4AB, which spans the NS4A and NS4B junction region. The secretion of SEAP into culture medium has been shown to depend on the cleavage of delta4AB by HCV NS3/4A protease. It is demonstrated that the amount of NS3/4A in Ava5-EG(delta4AB)SEAP cells correlated well with the copy numbers of HCV subgenomic RNA. It is also shown that replication of HCV subgenomic RNA inside cells is reflected by the alkaline phosphatase (SEAP) levels in culture medium. SEAP activity in the culture medium of Ava5-EG(delta4AB)SEAP was approximately 50-fold higher than the parental Ava5 cells. Ava5-EG(delta4AB)SEAP was validated as a drug screening system since several known HCV inhibitors were shown to reduce SEAP activities in culture media of Ava5-EG(delta4AB)SEAP cells. In conclusion, Ava5-EG(delta4AB)SEAP cells can be used to monitor HCV sub-genomic replication and the assay can be readily adapted to high throughput screening format to identify prospective anti-HCV drugs.

A prospective study characterizing full-length hepatitis B virus genomes during acute exacerbation.

BACKGROUND & AIMS: Hepatitis B virus (HBV) evolves rapidly in patients with chronic hepatitis B, and HBV variation may trigger acute exacerbation. To study this relationship, we investigated full-length viral sequences before, during, and after exacerbation. METHODS: We prospectively studied 14 patients with exacerbation of hepatitis B, either spontaneously (n = 4) or after receiving various medical interventions (n = 10), and measured their serum alanine aminotransferase (ALT) and HBV DNA levels monthly. Full-length HBV genomes at baseline, at the peak of serum viral load, at ALT peak, and after ALT peak were obtained by polymerase chain reaction, sequenced, and compared. Replication activities of serial HBV variants were assayed by in vitro transfection. RESULTS: Serum viral load was increased in all exacerbations. Viral peak preceded ALT peak in 13 (93%) of the 14 patients. At virologic peak, 12 patients (86%) harbored viral genome identical to the corresponding baseline genome. At and after ALT peak, 9 (64%) and 7 (50%) of the viral genomes remained identical to baseline, respectively. Mean nucleotide change per genome was 0.2 at virologic peak but increased to 4.4 and 8.1 at and after ALT peak, respectively. The replication potential of the viral variant that emerged during or after exacerbation was equivalent to that at baseline. CONCLUSIONS: Most exacerbations were preceded by an upsurge of serum HBV identical to the preexisting HBV strain. After exacerbation, about half of the patients were repopulated by a different viral variant, which was likely a result of immune selection.