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Objective: This study aimed to determine the effects of HBx on the inhibition of tumorigenesis in nude mice injected with CCL13-HBx cells. Therefore, the characteristics of the induced tumors and the phenomenon of apoptosis were assessed. Methods: The induced tumors were identified using the specific marker of hepatocellular carcinoma (HCC), anti-α-fetoprotein (AFP), and their characteristics were pathologically examined. Apoptosis was detected by DNA fragmentation, and the expression of the proapoptotic proteins p53, Bax, Bad, caspase-3, and caspase-8 and the anti-apoptotic protein Bcl-2 was detected by Western blotting. To identify possible molecules involved in the inhibition of tumorigenesis, extracts of the induced tumors were separated by 2D-PAGE, and the proteins were identified by MS. Results: The tumors of the nude mice injected with CCL13 and CCL13-HBx cells were identified as HCCs. Moreover, HBx was found to suppress tumor growth via apoptosis in the nude mice injected with CCL13-HBx cells. The MS findings revealed that phosphorylated myosin light chain was a candidate molecule involved in the inhibition of tumorigenesis. Conclusion: HBx suppressed tumorigenesis in the nude mice injected with CCL13-HBx cells, which proved to be a good animal model for the in vivo study of the effects of HBx on tumorigenesis. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Objective: The hepatitis B virus X protein (HBx) plays critical roles in cell survival via modulation of signaling pathways. In our previous studies, we reported that HBx inhibited the growth of CCL13-HBx-stable cells (Chang-HBx cells) in vitro and tumor formation in vivo in CCL13-HBx-cell-injected nude mice; however, this inhibition mechanism is unclear. Methods: To investigate the role of HBx in Wnt-3/β-catenin signaling pathways, we focused on the key molecules GSK-3β and β-catenin, and analyzed by Western blotting and immunofluorescence staining. Results: Results indicated that following HBx induction, GSK-3β activity was up-regulated, the expression and accumulation of β-catenin in the nucleus were decreased, and cell proliferation was suppressed. Inhibition of GSK-3β activity by pharmacological inhibitors rescued the expression and accumulation of β-catenin in the nucleus and facilitated cell proliferation and growth following HBx induction. The localization of β-catenin, which is involved in cell proliferation, and mediated by GSK-3β activation was also demonstrated. Conclusion: Our findings suggest that HBx negatively regulated proliferation of CCL13-HBx-stable cells via the GSK-3β/β-catenin cascade. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Objective: The known function of hepatitis B virus X protein (HBx) is to determine the fate of cells by modulating various signaling pathways. In our previous study, we demonstrated that HBx inhibits tumor formation in nude mice injected with CCL13-HBx stable cell lines; however, the mechanism underlying this inhibition is unclear. Methods: To investigate the possible mechanisms underlying HBx involvement in CCL13-HBx cells, gene profiles were initially analyzed by DNA microarray technology and subsequently confirmed by performing semiquantitative RT-PCR and Western blotting. Furthermore, the phenomenon of cell death via apoptosis was detected via DNA fragmentation, TUNEL staining, caspase-3 activity assay, and propidium iodide (PI) staining. Results: The results indicated that HBx induction downregulated Wnt-3 and β-catenin that are involved in cell proliferation. Moreover, HBx induction repressed cell growth and downregulated the expressions of cyclin D1, CDK4, cyclin E, CDK2, and cyclin B1. Furthermore, HBx induction triggered cell death via apoptosis, as determined by DNA fragmentation, TUNEL staining, caspase-3 activity assay, and PI staining. Conclusion: Most importantly, our results indicated that HBx induction in the CCL13-HBx stable cell line downregulated Wnt-3/β-catenin expression and suppressed cell growth by repressing cell proliferation or triggering cell apoptosis. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]