Modulation of MAPK pathways and cell cycle by replicating hepatitis B virus: Factors contributing to hepatocarcinogenesis☆
Introduction
The mechanism by which HBV results in HCC development is not well understood and the risk of developing HCC remains significantly elevated despite suppression of viral replication by antiviral therapy and after clearance of serum HBeAg and HBV viremia [1], [2], [3].
The HBx protein of HBV has been proposed as an important oncogenic stimulus for the development of HCC in chronically infected individuals [4], [5], [6], [7]. However several studies have reported divergent effects of HBx on p53-, Fas- and TNF-α-[8], [9], [10], c-myc-, and Ras-mediated apoptosis [11], [12], [13] and transactivation of IL-8 [14], [15], [16]. Our understanding of how HBV causes HCC remains a challenge and the frequent discrepancies in experimental findings may reflect limitations in duplicating cellular changes that occur in natural infection. The HBV large (LHB) and middle (MHB) envelope proteins have also been shown to contribute to hepatocarcinogenesis of HBV [17], [18], further emphasizing the role of viral factors, other than HBx, and active HBV replication as mediators of HCC development.
To address the role of active HBV replication on hepatocarcinogenesis we used a recombinant adenovirus system to deliver a replication competent HBV genome rather than single viral genes into primary marmoset hepatocytes, and contrasted the changes to cell signaling pathways induced by HBV infection with those seen in the human hepatoma cell line, Huh7.
Section snippets
Plasmids
A 1.5× full length replication competent HBV (genotype A, subtype adw2) [17] was subcloned into pAdTrack (provided by B Vogelstein, Howard Hughes Medical Centre, Baltimore) which was pre-digested with HindIII and EcoRV (Promega). The plasmid pAdTrack-HBVwt was digested with PmeI and transformed into AdEasier-1 cells by electroporation (Bio-Rad Gene Pulser). Clones (AdEasy-HBV) were transformed into Top 10F′ cells (Invitrogen) and confirmed by DNA sequencing. An AdEasy-GFP control expressing
HBV replication in Huh7 cells and PMH
Although Huh7 cells were known to be permissive for HBV replication this was not known for PMH. Infection of both Huh7 cells and PMH with rAdHBV resulted in expression of GFP in >95% of cells (Fig. 1a, b, e and f). Intra- and extra-cellular HBV DNA replication intermediates (relaxed circular, double stranded linear and single stranded) were readily detectable 72 h PI for Huh7, increasing thereafter until day 6 (Fig. 1c), and 24 h PI for PMH cells (Fig. 1g). HBV cccDNA was detected by quantitative
Discussion
Dysregulation of the signal transduction pathways such as Ras-MAPK signaling, IRS1/IGF pathways, NFkB, cell cycle, Wnt/β-catenin and apoptotic pathways have all been implicated in the development of HBV-associated carcinogenesis [7], [25], [26], [27], [28], [29], [30]. However, it is difficult to derive a unifying theme to explain the precise mechanism(s) linking the initiating oncogenic stimulus with subsequent changes in signal transduction. We sought to examine how HBV infection, rather than
Acknowledgements
The AdEasy system was generously provided by B Vogelstein (Howard Hughes Medical Institute, Baltimore). We thank Dr Scott Bowden and Ms Kathy Jackson, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia, for kindly performing quantitative real time PCR analyses for HBV cccDNA. This work was supported by grants of the Deutsche Krebshilfe, “Dr. Mildred-Scheel-Stiftung fuer Krebsforschung”, Grant No. 10-2142-Bo1.
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The authors who have taken part in this study declared that they have no relationship with the manufacturers of the product involved either in the past or present and did not receive funding from the manufacturers to carry out their research.