Blocking of G1/S transition and cell death in the regenerating liver of Hepatitis B virus X protein transgenic mice

doi:10.1016/j.bbrc.2005.12.089

Biochemical and Biophysical Research Communications

Volume 340, Issue 3, 17 February 2006, Pages 916-928

https://doi.org/10.1016/j.bbrc.2005.12.089 Get rights and content

Abstract

The Hepatitis B virus X (HBx) protein has been strongly implicated in the carcinogenesis of hepatocellular carcinoma (HCC). However, effects of the HBx protein on cell proliferation and cell death are controversial. This study investigates the effects of the HBx protein on liver regeneration in two independent lines of HBx transgenic mice, which developed HCC at around 14 to 16 months of age. High mortality, lower liver mass restoration, and impaired liver regeneration were found in the HBx transgenic mice post-hepatectomy. The levels of alanine aminotransferase and α-fetoprotein detected post-hepatectomy increased significantly in the HBx transgenic livers, indicating that they were more susceptible to damage during the regenerative process. Prolonged activation of the immediate-early genes in the HBx transgenic livers suggested that the HBx protein creates a strong effect by promoting the transition of the quiescent hepatocytes from G0 to G1 phase. However, impaired DNA synthesis and mitosis, as well as inhibited activation of G1, S, and G2/M markers, were detected. These results indicated that HBx protein exerted strong growth arrest on hepatocytes and imbalanced cell-cycle progression resulting in the abnormal cell death; this was accompanied by severe fat accumulation and impaired glycogen storage in the HBx transgenic livers. In conclusion, this study provides the first physiological evidence that HBx protein blocks G1/S transition of the hepatocyte cell-cycle progression and causes both a failure of liver functionality and cell death in the regenerating liver of the HBx transgenic mice.

Section snippets

Materials and methods

The albumin-HBx transgenic construct. We have constructed a liver-specific transgenic vector, pAlb-In-pA-HS4, which contains the mouse albumin promoter (NCBI Accession No. J04738 bases 1–2376), a 0.33-kb synthetic intron, and a 0.28-kb bovine growth hormone poly(A) signal. Both the intron and poly(A) signal were derived from the pIRES-EGFP plasmid (Clontech No. 6064-1). Two direct repeats of chicken HS4 insulator (NCBI Accession No. U78775 bases 10–1199) were placed downstream of the poly(A)

Generation of HBx transgenic mice

The plasmid used to generate the HBx transgenic mice is shown in Fig. 1A. The albumin-HBx plasmid was constructed with HBx expression driven from the mouse albumin promoter and two copies of the chicken β-globin 5′HS4 insulators were used to protect it from positional effects [31]. Six founders with three males and three females of the albumin-HBx transgenic mice were generated by pronucleus microinjection of C57BL/6 fertilized eggs. Germline transmission of offspring with the HBx transgene was

Abnormal immediate-early gene expression, G1 arrest, and cell death in the regenerating livers of the HBx transgenic mice

About 70–90% of the HBx transgenic mice died after PH by day 3. Severe cell death was accompanied by abnormal metabolism and failure of liver functions, such as fat accumulation and decreased glycogen storage and this may explain the high mortality of the HBx transgenic mice post-hepatectomy. The impaired liver regeneration observed in the HBx transgenic lines could be the result of aberrant regulation of cell-cycle genes following hepatectomy. First we have shown that there was a prolonged

Acknowledgments

This work was supported by grants from the National Science Council (NRPGM 92GM064 and NSC93-2752-B-010-004-PAE) and the National Health Research Institute (MG-093-CP-06), Taiwan.

References (51)

W.N. Chen et al.
Expression of integrated hepatitis B virus X variants in human hepatocellular carcinomas and its significance
Biochem. Biophys. Res. Commun.
(2000)
D.Y. Yu et al.
Incidence of hepatocellular carcinoma in transgenic mice expressing the hepatitis B virus X-protein
J. Hepatol.
(1999)
Y. Lin et al.
Hepatitis B virus X protein is a transcriptional modulator that communicates with transcription factor IIB and the RNA polymerase II subunit 5
J. Biol. Chem.
(1997)
Y. Lin et al.
The hepatitis B virus X protein is a co-activator of activated transcription that modulates the transcription machinery and distal binding activators
J. Biol. Chem.
(1998)
K. Gottlob et al.
The hepatitis B virus HBx protein inhibits caspase 3 activity
J. Biol. Chem.
(1998)
H. Kim et al.
X-gene product of hepatitis B virus induces apoptosis in liver cells
J. Biol. Chem.
(1998)
Y. Shirakata et al.
Hepatitis B virus X protein induces cell death by causing loss of mitochondrial membrane potential
J. Biol. Chem.
(2003)
M. Chami et al.
Caspase-dependent alterations of Ca²⁺ signaling in the induction of apoptosis by hepatitis B virus X protein
J. Biol. Chem.
(2003)
N. Fausto
Liver regeneration
J. Hepatol.
(2000)
W. Potts et al.
Chicken beta-globin 5′HS4 insulators function to reduce variability in transgenic founder mice
Biochem. Biophys. Res. Commun.
(2000)

F.V. Chisari et al.

Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice

Cell

(1989)

Y. Shan et al.

NMDA receptor overstimulation triggers a prolonged wave of immediate early gene expression: relationship to excitotoxicity

Exp. Neurol.

(1997)

H. Yamauchi et al.

Impaired liver regeneration after partial hepatectomy in db/db mice

Exp. Toxicol. Pathol.

(2003)

K.E. Behrns et al.

Hepatic steatosis as a potential risk factor for major hepatic resection

J. Gastrointest. Surg.

(1998)

K. Hakamada et al.

Sinusoidal flow block after warm ischemia in rats with diet-induced fatty liver

J. Surg. Res.

(1997)

P. Letteron et al.

Acute and chronic hepatic steatosis lead to in vivo lipid peroxidation in mice

J. Hepatol.

(1996)

B. Fromenty et al.

Impaired mitochondrial function in microvesicular steatosis

J. Hepatol.

(1997)

M.J. Bouchard et al.

The enigmatic X gene of hepatitis B virus

J. Virol.

(2004)

Y. Wang et al.

Characterization of HBV integrants in 14 hepatocellular carcinomas: association of truncated X gene and hepatocellular carcinogenesis

Oncogene

(2004)

G.Y. Hwang et al.

Detection of the hepatitis B virus X protein (HBx) antigen and anti-HBx antibodies in cases of human hepatocellular carcinoma

J. Clin. Microbiol.

(2003)

C.M. Kim et al.

HBx gene of hepatitis B virus induces liver cancer in transgenic mice

Nature

(1991)

K. Koike et al.

High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice

Hepatology

(1994)

J. Benn et al.

Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade

Proc. Natl. Acad. Sci. USA

(1994)

J. Benn et al.

Hepatitis B virus HBx protein induces transcription factor AP-1 by activation of extracellular signal-regulated and c-Jun N-terminal mitogen-activated protein kinases

J. Virol.

(1996)

K. Koike et al.

Induction of cell cycle progression by hepatitis B virus HBx gene expression in quiescent mouse fibroblasts

J. Clin. Invest.

(1994)

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Blocking of G1/S transition and cell death in the regenerating liver of Hepatitis B virus X protein transgenic mice

Abstract

Section snippets

Materials and methods

Generation of HBx transgenic mice

Abnormal immediate-early gene expression, G1 arrest, and cell death in the regenerating livers of the HBx transgenic mice

Acknowledgments

Biochem. Biophys. Res. Commun.

J. Hepatol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Hepatol.

Biochem. Biophys. Res. Commun.

Cell

Exp. Neurol.

Exp. Toxicol. Pathol.

J. Gastrointest. Surg.

J. Surg. Res.

J. Hepatol.

J. Hepatol.

The enigmatic X gene of hepatitis B virus

J. Virol.

Characterization of HBV integrants in 14 hepatocellular carcinomas: association of truncated X gene and hepatocellular carcinogenesis

Oncogene

Detection of the hepatitis B virus X protein (HBx) antigen and anti-HBx antibodies in cases of human hepatocellular carcinoma

J. Clin. Microbiol.

HBx gene of hepatitis B virus induces liver cancer in transgenic mice

Nature

High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice

Hepatology

Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade

Proc. Natl. Acad. Sci. USA

Hepatitis B virus HBx protein induces transcription factor AP-1 by activation of extracellular signal-regulated and c-Jun N-terminal mitogen-activated protein kinases

J. Virol.

Induction of cell cycle progression by hepatitis B virus HBx gene expression in quiescent mouse fibroblasts

J. Clin. Invest.