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Choudhary HB, Mandlik SK, Mandlik DS. Role of p53 suppression in the pathogenesis of hepatocellular carcinoma. World J Gastrointest Pathophysiol 2023; 14:46-70. [PMID: 37304923 PMCID: PMC10251250 DOI: 10.4291/wjgp.v14.i3.46] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Revised: 05/19/2023] [Accepted: 05/31/2023] [Indexed: 06/01/2023] Open
Abstract
In the world, hepatocellular carcinoma (HCC) is among the top 10 most prevalent malignancies. HCC formation has indeed been linked to numerous etiological factors, including alcohol usage, hepatitis viruses and liver cirrhosis. Among the most prevalent defects in a wide range of tumours, notably HCC, is the silencing of the p53 tumour suppressor gene. The control of the cell cycle and the preservation of gene function are both critically important functions of p53. In order to pinpoint the core mechanisms of HCC and find more efficient treatments, molecular research employing HCC tissues has been the main focus. Stimulated p53 triggers necessary reactions that achieve cell cycle arrest, genetic stability, DNA repair and the elimination of DNA-damaged cells’ responses to biological stressors (like oncogenes or DNA damage). To the contrary hand, the oncogene protein of the murine double minute 2 (MDM2) is a significant biological inhibitor of p53. MDM2 causes p53 protein degradation, which in turn adversely controls p53 function. Despite carrying wt-p53, the majority of HCCs show abnormalities in the p53-expressed apoptotic pathway. High p53 in-vivo expression might have two clinical impacts on HCC: (1) Increased levels of exogenous p53 protein cause tumour cells to undergo apoptosis by preventing cell growth through a number of biological pathways; and (2) Exogenous p53 makes HCC susceptible to various anticancer drugs. This review describes the functions and primary mechanisms of p53 in pathological mechanism, chemoresistance and therapeutic mechanisms of HCC.
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Affiliation(s)
- Heena B Choudhary
- Department of Pharmacology, BVDU, Poona College of Pharmacy, Pune 411038, Maharashtra, India
| | - Satish K Mandlik
- Department of Pharmaceutics, BVDU, Poona College of Pharmacy, Pune 411038, Maharashtra, India
| | - Deepa S Mandlik
- Department of Pharmacology, BVDU, Poona College of Pharmacy, Pune 411038, Maharashtra, India
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Downregulation of miR-200a-3p induced by hepatitis B Virus X (HBx) Protein promotes cell proliferation and invasion in HBV-infection-associated hepatocarcinoma. Pathol Res Pract 2017; 213:1464-1469. [PMID: 29103765 DOI: 10.1016/j.prp.2017.10.020] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/24/2017] [Revised: 10/18/2017] [Accepted: 10/19/2017] [Indexed: 12/11/2022]
Abstract
BACKGROUND Hepatitis B Virus X (HBx) Protein encoded by HBV is believed to be the major player in the process of HBV-induced oncogenesis. Ectopic expression of miR-200a-3p was reported to be associated with diverse tumorigenesis. This study aimed to better understand the role of miR-200a-3p and its correlation with HBx in HBV-induced hepatocellular carcinoma (HCC). METHODS In this report, we examined the gene expression using quantitative RT-PCR and protein expression using Western blotting analysis. Cells were transfected with miR-200a-3p mimics or empty vector, and HBx-carrying vector or empty vector. Cell viability was tested using CCK-8 assay. Wound healing assay was performed to assess cell migration while Transwell assay was performed to evaluate cell invasion. RESULTS miR-200a-3p was downregulated in HBV-positive tissue samples compared with HBV-negative tissue samples. This result was further confirmed with HBV-positive and - negative cell lines. HBx protein was overexpressed in HBV-positive cells where expression of miR-200a-3p was significantly suppressed. Increased cell viability, altered cell cycle progression, increased cell migration and invasion occurred in HBx-overexpressed cells compared to its controls. In forced expressed miR-200a-3p cells, cell viability, cell migration and invasion were significantly decreased, and cell cycle status was altered compared to its controls. CONCLUSIONS Taken together, pathogenetic function of HBx is negatively correlated with miR-200a-3p in HBV-cased HCC through regulating cell viability, cell cycle arrest, cell migration and cell invasion.
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Wang Z, Wu Z, Huang P. The function of miRNAs in hepatocarcinogenesis induced by hepatitis B virus X protein. Oncol Rep 2017. [DOI: 10.3892/or.2017.5716] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
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Abstract
Liver cancer remains one of the most common human cancers with a high mortality rate. Therapies for hepatocellular carcinoma (HCC) remain ineffective, due to the heterogeneity of HCC with regard to both the etiology and mutation spectrum, as well as its chemotherapy resistant nature; thus surgical resection and liver transplantation remain the gold standard of patient care. The most common etiologies of HCC are extrinsic factors. Humans have multiple defense mechanisms against extrinsic factor-induced carcinogenesis, of which tumor suppressors play crucial roles in preventing normal cells from becoming cancerous. The tumor suppressor p53 is one of the most frequently mutated genes in liver cancer. p53 regulates expression of genes involved in cell cycle progression, cell death, and cellular metabolism to avert tumor development due to carcinogens. This review article mainly summarizes extrinsic factors that induce liver cancer and potentially have etiological association with p53, including aflatoxin B1, vinyl chloride, non-alcoholic fatty liver disease, iron overload, and infection of hepatitis viruses.
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Affiliation(s)
- Tim Link
- Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Tomoo Iwakuma
- Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA
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5
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Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx. Proc Natl Acad Sci U S A 2016; 113:2074-9. [PMID: 26858413 DOI: 10.1073/pnas.1525616113] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
HBx is a hepatitis B virus protein that is required for viral infectivity and replication. Anti-apoptotic Bcl-2 family members are thought to be among the important host targets of HBx. However, the structure and function of HBx are poorly understood and the molecular mechanism of HBx-induced carcinogenesis remains unknown. In this study, we report biochemical and structural characterization of HBx. The recombinant HBx protein contains metal ions, in particular iron and zinc. A BH3-like motif in HBx (residues 110-135) binds Bcl-2 with a dissociation constant of ∼193 μM, which is drastically lower than that for a canonical BH3 motif from Bim or Bad. Structural analysis reveals that, similar to other BH3 motifs, the BH3-like motif of HBx adopts an amphipathic α-helix and binds the conserved BH3-binding groove on Bcl-2. Unlike the helical Bim or Bad BH3 motif, the C-terminal portion of the bound HBx BH3-like motif has an extended conformation and makes considerably fewer interactions with Bcl-2. These observations suggest that HBx may modulate Bcl-2 function in a way that is different from that of the classical BH3-only proteins.
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6
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Niller HH, Ay E, Banati F, Demcsák A, Takacs M, Minarovits J. Wild type HBx and truncated HBx: Pleiotropic regulators driving sequential genetic and epigenetic steps of hepatocarcinogenesis and progression of HBV-associated neoplasms. Rev Med Virol 2015; 26:57-73. [PMID: 26593760 DOI: 10.1002/rmv.1864] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2015] [Revised: 09/30/2015] [Accepted: 10/15/2015] [Indexed: 12/23/2022]
Abstract
Hepatitis B virus (HBV) is one of the causative agents of hepatocellular carcinoma. The molecular mechanisms of tumorigenesis are complex. One of the host factors involved is apparently the long-lasting inflammatory reaction which accompanies chronic HBV infection. Although HBV lacks a typical viral oncogene, the HBx gene encoding a pleiotropic regulatory protein emerged as a major player in liver carcinogenesis. Here we review the tumorigenic functions of HBx with an emphasis on wild type and truncated HBx variants, and their role in the transcriptional dysregulation and epigenetic reprogramming of the host cell genome. We suggest that HBx acquired by the HBV genome during evolution acts like a cellular proto-onc gene that is activated by deletion during hepatocarcinogenesis. The resulting viral oncogene (v-onc gene) codes for a truncated HBx protein that facilitates tumor progression. Copyright © 2015 John Wiley & Sons, Ltd.
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Affiliation(s)
- Hans Helmut Niller
- Institute for Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany
| | - Eva Ay
- Department of Retrovirology, National Center for Epidemiology, Budapest, Hungary
| | - Ferenc Banati
- RT-Europe Nonprofit Research Center, Mosonmagyarovar, Hungary
| | - Anett Demcsák
- University of Szeged, Faculty of Dentistry, Department of Oral Biology and Experimental Dental Research, Szeged, Hungary
| | - Maria Takacs
- Division of Virology, National Center for Epidemiology, Budapest, Hungary
| | - Janos Minarovits
- University of Szeged, Faculty of Dentistry, Department of Oral Biology and Experimental Dental Research, Szeged, Hungary
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7
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Nault JC. Pathogenesis of hepatocellular carcinoma according to aetiology. Best Pract Res Clin Gastroenterol 2014; 28:937-47. [PMID: 25260319 DOI: 10.1016/j.bpg.2014.08.006] [Citation(s) in RCA: 57] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2014] [Accepted: 08/15/2014] [Indexed: 01/31/2023]
Abstract
Hepatocellular carcinoma is related to various etiologies including hepatitis B, hepatitis C, high alcohol intake, aflatoxin B1 and metabolic syndrome. Most of the time HCC developed on cirrhosis. Consequently, the mechanisms of carcinogenesis of these different risk factors are difficult to separate from the events leading to cirrhosis. In contrast, aflatoxin B1 and hepatitis B have a clear direct oncogenic role through point mutations in the TP53 tumour suppressor gene and insertional mutagenesis respectively. Finally, next-generation sequencing and transcriptome analysis will refine our knowledge of the relationship between aetiology and the genetic events that draw the mutational landscape of hepatocellular carcinoma.
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Affiliation(s)
- Jean-Charles Nault
- Inserm, UMR-1162, Génomique fonctionnelle des Tumeurs solides, IUH, Paris, F-75010, France; Université Paris Descartes, Labex Immuno-Oncology, Sorbonne Paris Cité, Faculté de Médecine, Paris, France; Service d'Hépatologie, Hôpital Jean Verdier, AP-HP, Bondy, France; Université Paris 13, Bobigny, France.
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8
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Xie N, Chen X, Zhang T, Liu B, Huang C. Using proteomics to identify the HBx interactome in hepatitis B virus: how can this inform the clinic? Expert Rev Proteomics 2013; 11:59-74. [PMID: 24308553 DOI: 10.1586/14789450.2014.861745] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Hepatitis B virus (HBV) is a small and enveloped DNA virus, of which chronic infection is the main risk factor of liver cirrhosis and hepatocellular carcinoma. Hepatitis B virus X protein (HBx) is a multifunctional protein encoded by HBV genome, which have significant effects on HBV replication and pathogenesis. Through directly interacting with cellular proteins, HBx is capable to promote HBV replication, regulate transcription of host genes, disrupt protein degradation, modulate signaling pathway, manipulate cell death and deregulate cell cycle. In this review, we briefly discuss the diversified effects of HBx-interactome and their potential clinical significances.
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Affiliation(s)
- Na Xie
- The State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, 610041, P.R. China
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9
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ECHS1 acts as a novel HBsAg-binding protein enhancing apoptosis through the mitochondrial pathway in HepG2 cells. Cancer Lett 2012. [PMID: 23178449 DOI: 10.1016/j.canlet.2012.11.030] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
We aimed to confirm the role of ECHS1 as a binding protein of HBsAg (HBs) and investigate its function during the development of hepatocellular carcinoma (HCC). Our results show that both exogenous and endogenous ECHS1 proteins bind to HBs and co-localize in the cytoplasm in vitro. The coexistence of HBs and ECHS1 enhances HepG2 cell apoptosis, affects ECHS1 localization in the mitochondria and induces apoptosis by decreasing the mitochondrial membrane potential (MMP). These findings suggest that ECHS1 may be applied as a potential therapeutic target during the treatment of HBV-related hepatitis or HCC.
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10
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Mason WS. Hepadnaviruses and Hepatocellular Carcinoma. CANCER ASSOCIATED VIRUSES 2012:531-569. [DOI: 10.1007/978-1-4614-0016-5_22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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11
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Katiyar S, Jiao X, Addya S, Ertel A, Covarrubias Y, Rose V, Casimiro MC, Zhou J, Lisanti MP, Nasim T, Fortina P, Pestell RG. Mammary gland selective excision of c-jun identifies its role in mRNA splicing. Cancer Res 2011; 72:1023-34. [PMID: 22174367 DOI: 10.1158/0008-5472.can-11-3647] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The c-jun gene regulates cellular proliferation and apoptosis via direct regulation of cellular gene expression. Alternative splicing of pre-mRNA increases the diversity of protein functions, and alternate splicing events occur in tumors. Here, by targeting the excision of the endogenous c-jun gene within the mouse mammary epithelium, we have identified its selective role as an inhibitor of RNA splicing. Microarray-based assessment of gene expression, on laser capture microdissected c-jun(-/-) mammary epithelium, showed that endogenous c-jun regulates the expression of approximately 50 genes governing RNA splicing. In addition, genome-wide splicing arrays showed that endogenous c-jun regulated the alternate exon of approximately 147 genes, and 18% of these were either alternatively spliced in human tumors or involved in apoptosis. Endogenous c-jun also was shown to reduce splicing activity, which required the c-jun dimerization domain. Together, our findings suggest that c-jun directly attenuates RNA splicing efficiency, which may be of broad biologic importance as alternative splicing plays an important role in both cancer development and therapy resistance.
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Affiliation(s)
- Sanjay Katiyar
- Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
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Martin-Vilchez S, Lara-Pezzi E, Trapero-Marugán M, Moreno-Otero R, Sanz-Cameno P. The molecular and pathophysiological implications of hepatitis B X antigen in chronic hepatitis B virus infection. Rev Med Virol 2011; 21:315-29. [PMID: 21755567 DOI: 10.1002/rmv.699] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2011] [Revised: 05/23/2011] [Accepted: 05/26/2011] [Indexed: 12/16/2022]
Abstract
Hepatitis B virus is considered one of the most significant environmental carcinogens in humans. Because the mechanisms of HBV replication and the development of hepatocellular carcinoma (HCC) are partially known, HBV-associated pathogenesis remains a challenge to increase its understanding. Evidence suggests that the regulatory protein hepatitis B virus X (HBx) mediates the establishment and maintenance of the chronic carrier state. HBx is a multifunctional and potentially oncogenic protein that is conserved among mammalian hepadnaviruses; it is produced very early after infection and throughout the chronic phase. HBx exerts its effects by interacting with cellular proteins and activating various signaling pathways. HBx stimulates the transcription of genes that regulate cell growth, apoptosis, and DNA repair. It also interacts with proteasome subunits and affects mitochondrial stability. Moreover, HBx participates in processes that are associated with the progression of chronic liver disease, including angiogenesis and fibrosis. This review discusses the function of HBx in the life cycle of HBV and its contribution to the pathogenesis of HCC.
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Affiliation(s)
- Samuel Martin-Vilchez
- CIBERehd, ISCIII, Madrid, Spain; Servicio Digestivo, Hospital Universitario "La Princesa" and Instituto de Investigación Biomédica "La Princesa", Madrid, Spain
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13
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Kim KH. [Pro-apoptotic function of hepatitis B virus X protein]. THE KOREAN JOURNAL OF HEPATOLOGY 2010; 16:112-22. [PMID: 20606495 DOI: 10.3350/kjhep.2010.16.2.112] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Infection of hepatitis B virus (HBV) is a main cause of liver diseases including hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Among the HBV-encoded proteins, the HBV X protein (HBx) has been suspected to be strongly involved in HBV-associated liver pathogenesis. HBx, a virally encoded multifunctional regulator, has been shown to induce apoptosis, anti-apoptosis, proliferation, and transformation of cells depending on the cell lines, model systems used, assay protocols, and research groups. Among the several activities of HBx, the pro-apoptotic function of HBx will be discussed in this review. Given that the disruption of apoptosis pathway by HBx contributes to the liver pathogenesis, a better understanding of the molecular interference in the cellular pro-apoptotic networks by HBx will provide useful clues for the intervention in HBV-mediated liver diseases.
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Affiliation(s)
- Kyun-Hwan Kim
- Department of Pharmacology, School of Medicine and Center for Cancer Research and Diagnostic Medicine, IBST, Konkuk University, Seoul, Korea.
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14
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Redox regulation of T-cell turnover by the p13 protein of human T-cell leukemia virus type 1: distinct effects in primary versus transformed cells. Blood 2010; 116:54-62. [DOI: 10.1182/blood-2009-07-235861] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
AbstractThe present study investigated the function of p13, a mitochondrial protein of human T-cell leukemia virus type 1 (HTLV-1). Although necessary for viral propagation in vivo, the mechanism of function of p13 is incompletely understood. Drawing from studies in isolated mitochondria, we analyzed the effects of p13 on mitochondrial reactive oxygen species (ROS) in transformed and primary T cells. In transformed cells (Jurkat, HeLa), p13 did not affect ROS unless the cells were subjected to glucose deprivation, which led to a p13-dependent increase in ROS and cell death. Using RNA interference we confirmed that expression of p13 also influences glucose starvation-induced cell death in the context of HTLV-1–infected cells. ROS measurements showed an increasing gradient from resting to mitogen-activated primary T cells to transformed T cells (Jurkat). Expression of p13 in primary T cells resulted in their activation, an effect that was abrogated by ROS scavengers. These findings suggest that p13 may have a distinct impact on cell turnover depending on the inherent ROS levels; in the context of the HTLV-1 propagation strategy, p13 could increase the pool of “normal” infected cells while culling cells acquiring a transformed phenotype, thus favoring lifelong persistence of the virus in the host.
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15
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Neuveut C, Wei Y, Buendia MA. Mechanisms of HBV-related hepatocarcinogenesis. J Hepatol 2010; 52:594-604. [PMID: 20185200 DOI: 10.1016/j.jhep.2009.10.033] [Citation(s) in RCA: 335] [Impact Index Per Article: 22.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/22/2009] [Revised: 10/22/2009] [Accepted: 10/23/2009] [Indexed: 12/04/2022]
Abstract
The hepatitis B virus (HBV) is a small enveloped DNA virus, which primarily infects hepatocytes and causes acute and persistent liver disease. Epidemiological studies have provided overwhelming evidence for a causal role of chronic HBV infection in the development of hepatocellular carcinoma, but the molecular mechanisms underlying virally-induced tumourigenesis remain largely debated. In the absence of a dominant oncogene encoded by the HBV genome, indirect roles have been proposed, including insertional activation of cellular cancer-related genes by HBV DNA integration, induction of genetic instability by viral integration or by the regulatory protein HBx, and long-term effects of viral proteins in enhancing immune-mediated liver disease. Recent genetic studies indicate that HBV-related tumours display a distinctive profile with a high rate of chromosomal alterations and low frequency of beta-catenin mutations. This review will discuss the evidence implicating chronic HBV infection as a causal risk factor of primary liver cancer. It will also discuss the molecular mechanisms that are critical for the tumourigenic process due to long lasting infection with HBV.
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Affiliation(s)
- Christine Neuveut
- Oncogenesis and Molecular Virology Unit, Institut Pasteur, Inserm U579, 28 rue du Dr Roux, Paris cedex 15, France
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16
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Liver cell transformation in chronic HBV infection. Viruses 2009; 1:630-646. [PMID: 21994562 PMCID: PMC3185520 DOI: 10.3390/v1030630] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2009] [Revised: 10/26/2009] [Accepted: 10/29/2009] [Indexed: 12/20/2022] Open
Abstract
Epidemiological studies have provided overwhelming evidence for a causal role of chronic HBV infection in the development of hepatocellular carcinoma (HCC), but the molecular mechanisms underlying virally-induced tumorigenesis remain largely debated. In the absence of a dominant oncogene encoded by the HBV genome, indirect roles have been proposed, including insertional activation of cellular oncogenes by HBV DNA integration, induction of genetic instability by viral integration or by the regulatory protein HBx, and long term effects of viral proteins in enhancing immune-mediated liver disease. In this chapter, we discuss different models of HBV-mediated liver cell transformation based on animal systems of hepadnavirus infection as well as functional studies in hepatocyte and hepatoma cell lines. These studies might help identifying the cellular effectors connecting HBV infection and liver cell transformation.
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Sahdev S, Saini KS, Hasnain SE. Baculovirus P35 protein: An overview of its applications across multiple therapeutic and biotechnological arenas. Biotechnol Prog 2009; 26:301-12. [DOI: 10.1002/btpr.339] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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Abstract
There are eight genotypes of hepatitis B virus (HBV). Most genotypes can be further divided into subgenotypes. HBV genotypes influence the course of disease and treatment, and show geographic preferences. In Europe, A and D are the main genotypes found. Genotype A is more prevalent in northern Europe, where genotype D is mainly found in countries surrounding the Mediterranean Sea and in Eastern Europe. Subgenotype A2 is the dominant subgenotype in Europe, but the geographic prevalence of the four subgenotypes found in genotype D is not yet clear. On treatment with interferon HBV, genotype A, compared to genotype D, showsbetter virological response and in a large proportion of patients, even development of anti-HBs. However, in the first year of treatment with lamivudine, higher rates of emergence of YMDD variants are observed in genotype A. This work summarizes the current knowledge on HBV genotypes in countries with low and intermediate HBV carriership from Europe.
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Affiliation(s)
- Stephan Schaefer
- Department of Virology, Institute for Medical Microbiology, Virology and Hygiene, Rostock University, Rostock, Germany
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20
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Rizzetto M, Zoulim F. Viral Hepatitis. TEXTBOOK OF HEPATOLOGY 2007:819-956. [DOI: 10.1002/9780470691861.ch9a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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Abstract
Irrespective of the morphological features of end-stage cell death (that may be apoptotic, necrotic, autophagic, or mitotic), mitochondrial membrane permeabilization (MMP) is frequently the decisive event that delimits the frontier between survival and death. Thus mitochondrial membranes constitute the battleground on which opposing signals combat to seal the cell's fate. Local players that determine the propensity to MMP include the pro- and antiapoptotic members of the Bcl-2 family, proteins from the mitochondrialpermeability transition pore complex, as well as a plethora of interacting partners including mitochondrial lipids. Intermediate metabolites, redox processes, sphingolipids, ion gradients, transcription factors, as well as kinases and phosphatases link lethal and vital signals emanating from distinct subcellular compartments to mitochondria. Thus mitochondria integrate a variety of proapoptotic signals. Once MMP has been induced, it causes the release of catabolic hydrolases and activators of such enzymes (including those of caspases) from mitochondria. These catabolic enzymes as well as the cessation of the bioenergetic and redox functions of mitochondria finally lead to cell death, meaning that mitochondria coordinate the late stage of cellular demise. Pathological cell death induced by ischemia/reperfusion, intoxication with xenobiotics, neurodegenerative diseases, or viral infection also relies on MMP as a critical event. The inhibition of MMP constitutes an important strategy for the pharmaceutical prevention of unwarranted cell death. Conversely, induction of MMP in tumor cells constitutes the goal of anticancer chemotherapy.
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Affiliation(s)
- Guido Kroemer
- Institut Gustave Roussy, Institut National de la Santé et de la Recherche Médicale Unit "Apoptosis, Cancer and Immunity," Université de Paris-Sud XI, Villejuif, France
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Cheng ASL, Wong N, Tse AMY, Chan KYY, Chan KK, Sung JJY, Chan HLY. RNA interference targeting HBx suppresses tumor growth and enhances cisplatin chemosensitivity in human hepatocellular carcinoma. Cancer Lett 2007; 253:43-52. [PMID: 17296261 DOI: 10.1016/j.canlet.2007.01.004] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2006] [Revised: 12/31/2006] [Accepted: 01/05/2007] [Indexed: 12/22/2022]
Abstract
The X protein of hepatitis B virus (HBx) is often expressed in human hepatocellular carcinoma (HCC) but its role on tumor growth is not fully clarified. In this study, RNA interference was employed to knockdown HBx expression in Hep3B HCC cells, which naturally express carboxyl-end truncated form of HBx frequently found in HCC tissues. Specific knockdown of HBx strongly inhibited cell growth and tumorigenicity in xenograft model. HBx repression induced apoptosis in Hep3B cells and significantly increased cell sensitivity to cisplatin-induced apoptosis. These results suggest that RNA interference-mediated HBx suppression exerts potent anti-proliferative and chemosensitizing effects in human HCC.
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Affiliation(s)
- Alfred S L Cheng
- Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, Hong Kong
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Ping-Yuan L, Hung-Jen L, Meng-Jiun L, Feng-Ling Y, Hsue-Yin H, Jeng-Woei L, Wen-Ling S. Avian Reovirus activates a novel proapoptotic signal by linking Src to p53. Apoptosis 2006; 11:2179-93. [PMID: 17051326 DOI: 10.1007/s10495-006-0291-6] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
We have previously shown that avian reovirus (ARV) S1133 and its structural protein sigmaC cause apoptosis in cultured Vero cells through an unknown intracellular signaling pathway. This work investigates how ARV S1133 induces proapoptotic signals. Upon ARV S1133 infection and subsequent apoptosis, levels of p53 mRNA and protein, and p53 serine-46 and serine-392 phosphorylation increased. In addition, p53-driven reporter activity and levels of the p53-induced apoptotic protein bax were increased, and Src tyrosine-418 phosphorylation was elevated. UV-inactivated virus failed to activate Src, p53 or induce apoptosis. Over-expression of dominant negative p53, or treatment with tyrosine kinase inhibitor genistein protected cells from ARV S1133-induced apoptosis. Inhibition of Src by over-expression of C-terminal Src kinase (Csk) or treatment with Src family tyrosine kinase inhibitor SU-6656 diminished the ARV S1133-induced p53 expression, activation, and apoptosis. Over-expression of sigmaC resulted in the upregulation of p53, p53 serine-46 phosphorylation, p53-driven reporter activity and accumulation of bax. sigmaC expression during ARV S1133 infection was concomitant with the onset of apoptosis. These studies provide strong evidence that the viral gene expression is required for ARV S1133 to initiate a proapoptotic signal via Src to p53. In addition, sigmaC was able to utilize a p53-dependent pathway to elicit apoptosis.
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Affiliation(s)
- Lin Ping-Yuan
- Graduate Institute and Department of Life Science, Tzu-Chi University, 701, Sec. 3, Chung-Yang Rd., Hualien, Taiwan, 970
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Fischer SF, Schmidt K, Fiedler N, Glebe D, Schüttler C, Sun J, Gerlich WH, Repp R, Schaefer S. Genotype-dependent activation or repression of HBV enhancer II by transcription factor COUP-TF1. World J Gastroenterol 2006; 12:6054-8. [PMID: 17009409 PMCID: PMC4124418 DOI: 10.3748/wjg.v12.i37.6054] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/05/2006] [Revised: 05/28/2006] [Accepted: 06/14/2006] [Indexed: 02/06/2023] Open
Abstract
AIM To study the expression of HBV enhancer II by transcription factor COUP-TF1. METHODS In order to study the regulation of HBV variants in the vicinity of the NRRE we cloned luciferase constructs containing the HBV enhancer II from variants and from HBV genotypes A and D and cotransfected them together with expression vectors for COUP-TF1 into HepG2 cells. RESULTS Our findings show that enhancer II of HBV genotype A is also repressed by COUP-TF1. In contrast, two different enhancer II constructs of HBV genotype D were activated by COUP-TF1. The activation was independent of the NRRE because a natural variant with a deletion of nt 1763-1770 was still activated by COUP-TF1. CONCLUSION Regulation of transcription of the HBV genome seems to differ among HBV genomes derived from different genotypes. These differences in transcriptional control among HBV genotypes may be the molecular basis for differences in the clinical course among HBV genotypes.
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Affiliation(s)
- Silke F Fischer
- Institut für Medizinische Virologie, Justus-Liebig-Universität Giessen, Germany
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25
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Tang H, Oishi N, Kaneko S, Murakami S. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci 2006; 97:977-83. [PMID: 16984372 PMCID: PMC11159107 DOI: 10.1111/j.1349-7006.2006.00299.x] [Citation(s) in RCA: 242] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Chronic infection of hepatitis B virus (HBV) is one of the major causes of hepatocellular carcinoma (HCC) in the world. Hepatitis B virus X protein (HBx) has been long suspected to be involved in hepatocarcinogenesis, although its oncogenic role remains controversial. HBx is a multifunctional regulator that modulates transcription, signal transduction, cell cycle progress, protein degradation pathways, apoptosis, and genetic stability by directly or indirectly interacting with host factors. This review focuses on the biological roles of HBx in HBV replication and cellular transformation in terms of the molecular functions of HBx. Using the transient HBV replication assay, ectopically expressed HBx could stimulate HBV transcription and replication with the X-defective replicon to the level of those with the wild one. The transcription coactivation is mainly contributing to the stimulatory role of HBx on HBV replication although the other functions may affect HBV replication. Effect of HBx on cellular transformation remains controversial and was never addressed with human primary or immortal cells. Using the human immortalized primary cells, HBx was found to retain the ability to overcome active oncogene RAS-induced senescence that requires full-length HBx. At least two functions of HBx, the coactivation function and the ability to overcome oncogene-induced senescence, may be cooperatively involved in HBV-related hepatocarcinogenesis.
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Affiliation(s)
- Hong Tang
- Division of Biotherapy of Infectious Diseases, Key Laboratory of Biotherapy of Human Diseases, Ministry of Education, Chengdu, Sichuan, China
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26
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N/A, 林 纳. N/A. Shijie Huaren Xiaohua Zazhi 2006; 14:2579-2585. [DOI: 10.11569/wcjd.v14.i26.2579] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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Li H, Chi CY, Lee S, Andrisani OM. The mitogenic function of hepatitis B virus X protein resides within amino acids 51 to 140 and is modulated by N- and C-terminal regulatory regions. J Virol 2006; 80:10554-64. [PMID: 16920820 PMCID: PMC1641793 DOI: 10.1128/jvi.00661-06] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
The hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. pX variants encoded by HBV genomes found integrated in genomic DNA from liver tumors of patients with hepatocellular carcinoma (HCC) generally lack amino acids 134 to 154. Since deregulation of mitogenic pathways is linked to oncogenic transformation, herein we define the pX region required for mitogenic pathway activation. A series of pX deletions was used to construct tetracycline-regulated pX-expressing cell lines. The activation of the mitogenic pathways by these pX deletions expressed in the constructed cell lines was measured by transient transreporter assays, effects on endogenous cyclin A expression, and apoptosis. Conditional expression of pX51-140 in AML12 clone 4 cell line activates the mitogenic pathways, induces endogenous cyclin A expression, and sensitizes cells to apoptosis, similar to wild-type (WT) pX. By contrast, pX1-115 is inactive, supporting the idea that amino acids 116 to 140 are required for mitogenic pathway activation. Moreover, this pX deletion analysis demonstrates that WT pX function is modulated by two regions spanning amino acids 1 to 78 and 141 to 154. The N-terminal X1-78, expressed via a retroviral vector in WT pX-expressing 4pX-1 cells, coimmunoprecipitates with WT pX, indicating this pX region participates in protein-protein interactions leading to pX oligomerization. Interestingly, pX1-78 interferes with WT pX in mediating mitogenic pathway activation, endogenous gene expression, and apoptosis. The C-terminal pX region spanning amino acids 141 to 154 decreases pX stability, determined by pulse-chase studies of WT pX and pX1-140, suggesting that increased stability of naturally occurring pX variants lacking amino acids 134 to 154 may play a role in HCC development.
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Affiliation(s)
- Huajie Li
- Department of Basic Medical Sciences, Purdue University, 625 Harrison Street, West Lafayette, IN 47907-2026, USA
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28
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Fiedler N, Quant E, Fink L, Sun J, Schuster R, Gerlich WH, Schaefer S. Differential effects on apoptosis induction in hepatocyte lines by stable expression of hepatitis B virus X protein. World J Gastroenterol 2006; 12:4673-82. [PMID: 16937438 PMCID: PMC4087832 DOI: 10.3748/wjg.v12.i29.4673] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: Hepatitis B virus protein X (HBx) has been shown to be weakly oncogenic in vitro. The transforming activities of HBx have been linked with the inhibition of several functions of the tumor suppressor p53. We have studied whether HBx may have different effects on p53 depending on the cell type.
METHODS: We used the human hepatoma cell line HepG2 and the immortalized murine hepatocyte line AML12 and analyzed stably transfected clones which expressed physiological amounts of HBx. P53 was induced by UV irradiation.
RESULTS: The p53 induction by UV irradiation was unaffected by stable expression of HBx. However, the expression of the cyclin kinase inhibitor p21waf/cip/sdi which gets activated by p53 was affected in the HBx transformed cell line AML12-HBx9, but not in HepG2. In AML-HBx9 cells, p21waf/cip/sdi-protein expression and p21waf/cip/sdi transcription were deregulated. Furthermore, the process of apoptosis was affected in opposite ways in the two cell lines investigated. While stable expression of HBx enhanced apoptosis induced by UV irradiation in HepG2-cells, apoptosis was decreased in HBx transformed AML12-HBx9. P53 repressed transcription from the HBV enhancer I, when expressed from expression vectors or after induction of endogenous p53 by UV irradiation. Repression by endogenous p53 was partially reversible by stably expressed HBx in both cell lines.
CONCLUSION: Stable expression of HBx leads to deregulation of apoptosis induced by UV irradiation depending on the cell line used. In an immortalized hepatocyte line HBx acted anti-apoptotic whereas expression in a carcinoma derived hepatocyte line HBx enhanced apoptosis.
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Affiliation(s)
- Nicola Fiedler
- Abt. Virologie, Institut fur Medizinische Mikrobiologie, Schillingallee 70, Universitat Rostock, D-18055 Rostock, Germany
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29
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Wei W, Huang W, Pan Y, Zhu F, Wu J. Functional switch of viral protein HBx on cell apoptosis, transformation, and tumorigenesis in association with oncoprotein Ras. Cancer Lett 2006; 244:119-28. [PMID: 16569476 DOI: 10.1016/j.canlet.2005.12.008] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2005] [Revised: 11/26/2005] [Accepted: 12/05/2005] [Indexed: 01/17/2023]
Abstract
The X protein (HBx) of hepatitis B virus (HBV) plays important roles in hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) during viral infection. In this study, we demonstrated that co-transfection of mouse embryo fibroblasts (STO) with HBx and activated Ras triggered apoptotic cell death, while HBx or activated Ras individually failed to induce apoptosis. In addition, STO cells were able to form colonies on soft agar after transfected with HBx or Ras, and cells co-transfected with both genes failed to transform. Moreover, nude mice injected with STO cells carrying either HBx or Ras could develop tumor, but tumor growth was inhibited by the injection of both STO cells harboring HBx and carrying Ras. These results suggested that HBx plays a role as a tumor inducer and stimulates neoplastic transformation of normal cells, but shifts its function to the induction of apoptosis in association with Ras.
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Affiliation(s)
- Wei Wei
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, People's Republic of China
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30
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Lu YW, Chen WN. Human hepatitis B virus X protein induces apoptosis in HepG2 cells: Role of BH3 domain. Biochem Biophys Res Commun 2005; 338:1551-6. [PMID: 16274670 DOI: 10.1016/j.bbrc.2005.10.117] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2005] [Accepted: 10/19/2005] [Indexed: 11/25/2022]
Abstract
The smallest protein of hepatitis B virus, HBX, has been implicated in the development of liver diseases by interfering with normal cellular processes. Its role in cell proliferation has been unclear as both pro-apoptotic and anti-apoptotic activities have been reported. We showed molecular evidence that HBX induced apoptosis in HepG2 cells. A Bcl-2 Homology Domain 3 was identified in HBX, which interacted with anti-apoptotic but not pro-apoptotic members of the Bcl-2 family of proteins. HBX induced apoptosis when transfected into HepG2 cells, as demonstrated by both flow cytometry and caspase-3 activity. However, HBX protein may not be stable in apoptotic cells triggered by its own expression as only its mRNA or the fusion protein with the glutathione-S-transferase was detected in transfected cells. Our results suggested that HBX behaved as a pro-apoptotic protein and was able to induce apoptosis.
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Affiliation(s)
- Yi Wei Lu
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive 05N-10, Singapore 637551, Singapore
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31
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Abstract
AIM: To investigate the effect of hepatitis B virus (HBV) X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells.
METHODS: The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group.
RESULTS: HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferation capacity was obviously lower in HepG2/pcDNA3-X cells (0.08910±0.003164) than in HepG2 (0.14410±0.004927) and HepG2/pcDNA3 cells (0.12150±0.007159) (P<0.05 and P<0.01). Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells (980/2 000) than HepG2 (420/2 000), HepG2/pcDNA3 cells (520/2 000) (P<0.05 and P<0.01). Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/pcDNA3cells (P<0.05 and P<0.01). Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells (P<0.05 and P<0.01). Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells (P<0.05 andP<0.01).
CONCLUSION: HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.
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Affiliation(s)
- Na Lin
- Department of Gastroenterology, Affiliated Union Hospital, Fujian Medical University, Fuzhou 350001, Fujian Province, China
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32
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Buendia MA, Paterlini‐Bréchot P, Tiollais P, Bréchot C. Hepatocellular Carcinoma: Molecular Aspects in Hepatitis B. VIRAL HEPATITIS 2005:269-294. [DOI: 10.1002/9780470987131.ch17] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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Miao J, Chen GG, Chun SY, Lai PPS. Hepatitis B virus X protein induces apoptosis in hepatoma cells through inhibiting Bcl-xL expression. Cancer Lett 2005; 236:115-24. [PMID: 15990224 DOI: 10.1016/j.canlet.2005.05.014] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2005] [Revised: 05/04/2005] [Accepted: 05/06/2005] [Indexed: 02/06/2023]
Abstract
The X protein of hepatitis B virus (HBx) exhibits numerous activities affecting gene transcription, intracellular signal transduction, cell proliferation and apoptosis. Recent studies showed that HBx induced apoptosis by causing loss of mitochondrial membrane potential, suggesting that HBx-mediated apoptosis is mitochondria-dependent. However, the molecular mechanism of the gene in this pathway is still far from understood. In this study, we demonstrated that introduction of HBx into a hepatocellular carcinoma cell line, Hep3B, caused apoptosis and sensitized the cell to TNFalpha-induced cell killing. Over-expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein, prevented cell death dragged by HBx. Importantly, expression of HBx in Hep3B cells reduced Bcl-xL mRNA and protein levels, but did not regulate other Bcl-2 family members. Although, HBx itself did not affect intracellular distribution of cytochrome c, an enhanced release of cytochrome c from mitochondria was observed when TNFalpha was applied. Thus, the introduction of HBx into Hep3B cells induces apoptosis and sensitizes Hep3B cells to TNFalpha-mediated cell killing, and these processes may accomplish through inhibiting Bcl-xL expression and subsequently promoting cytochrome c release from mitochondria.
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Affiliation(s)
- Ji Miao
- Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong SAR
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34
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Kingsman SM, Mitrophanous K, Olsen JC. Potential oncogene activity of the woodchuck hepatitis post-transcriptional regulatory element (WPRE). Gene Ther 2004; 12:3-4. [PMID: 15510172 DOI: 10.1038/sj.gt.3302417] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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36
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Verrier F, Mignotte B, Jan G, Brenner C. Study of PTPC composition during apoptosis for identification of viral protein target. Ann N Y Acad Sci 2004; 1010:126-42. [PMID: 15033708 DOI: 10.1196/annals.1299.022] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
The permeability transition pore complex (PTPC), a mitochondrial polyprotein complex, has been previously described to be involved in the control of mitochondrial membrane permeabilization (MMP) during chemotherapy-induced apoptosis. PTPC may contain proteins from both mitochondrial membranes [e.g., voltage-dependent anion channel (VDAC), PRAX-1, peripheral benzodiazepine receptor (PBR), adenine nucleotide translocator (ANT)], from cytosol (e.g., hexokinase II, glycerol kinase), from matrix [e.g., cyclophilin D (CypD)], and from intermembrane space (e.g., creatine kinase). PTPC may also interact with tumor suppressor proteins (i.e., Bax and Bid), oncoprotein homologues of Bcl-2 and some viral proteins, which can regulate apoptosis induced by pore opening. ANT and VDAC are the target of numerous pro-apoptotic MMP inducers. However, the precise composition of PTPC as well as the respective role of each PTPC component represent major issues in the understanding MMP process. Using several experimental strategies that combine co-immunoprecipitation, proteomics, and functional tests with proteoliposomes, we and others have been able to characterize some of the intra/inter-PTPC protein interactions leading to a better understanding of the process of MMP. In addition, this approach could identify new putative members and regulators of PTPC pro-apoptotic function and new targets of viral protein involved in the modulation of apoptosis during infection.
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Affiliation(s)
- Florence Verrier
- CNRS FRE 2445, Université de Versailles/St. Quentin, 45, avenue des Etats-Unis, 78035 Versailles, France
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37
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Shih WL, Hsu HW, Liao MH, Lee LH, Liu HJ. Avian reovirus sigmaC protein induces apoptosis in cultured cells. Virology 2004; 321:65-74. [PMID: 15033566 DOI: 10.1016/j.virol.2003.12.004] [Citation(s) in RCA: 89] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2003] [Revised: 12/03/2003] [Accepted: 12/03/2003] [Indexed: 10/26/2022]
Abstract
The avian reovirus (ARV) infection is associated with various disease conditions in poultry. However, the pathogenesis mechanisms are poorly characterized. In the present study, we clearly demonstrated that the sigmaC of ARV S1133 strain induced apoptosis in both BHK-21 and Vero cells. Five kinds of assays for apoptosis were used in analyzing ARV-infected BHK-21 and Vero cells: (1) assay for DNA ladders, (2) ELISA detection of cytoplasmic histone-associated DNA fragments, (3) nuclear staining with acridine orange, (4) Western blot, Northern blot, and immunofluorescent assay (IFA), and (5) flow cytometric analysis. The sigmaC protein of ARV could elicit apoptosis occurring in a dose- and time-dependent manner. The current results further our understanding of the function of sigmaC in cultured cells and suggest that sigmaC is a viral-encoded apoptin and possesses apoptosis-inducing ability. Furthermore, deletion analysis of the ARV sigmaC protein suggests that the carboxyl-terminus of sigmaC is important in mediating sigmaC-induced apoptosis because its deletion abolished the induction of apoptosis.
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Affiliation(s)
- Wen L Shih
- Department of Life Science, Tzu-Chi University, Hualien, Taiwan
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38
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Chen HY, Tang NH, Li XJ, Zhang SJ, Chen ZX, Wang XZ. Transfection and expression of hepatitis B virus x gene and its effect on apoptosis in HL-7702 cells. World J Gastroenterol 2004; 10:959-64. [PMID: 15052674 PMCID: PMC4717112 DOI: 10.3748/wjg.v10.i7.959] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the effects of hepatitis B virus x gene and its protein product HBxAg on apoptosis in hepatocyte line HL-7702.
METHODS: The reconstituted plasmid pcDNA3-x was established through recombination DNA technique; pcDNA3-X was transfected into HL-7702 cells by lipid-mediated trasfection. Positive clones were screened by G418, and HL-7702/HBx cells were analysed by the RT-PCR to confirm the steady expression of X gene in HL-7702 cells. The apoptosis rate in HL-7702 cells was determined by flow cytometry, TUNEL technology, electronic microscope. At the mean time, pcDNA3-X was transfected transiently into HL-7702 cells, and total RNA from HL-7702 cells was extracted 24, 48, 72, 96 and 120 h after the transient transfection, and semi-quantitative analysis was performed by RT-PCR to detect the expression of HBV X gene. Furthermore, apoptosis rate in HL-7702 cells was determined by flow cytometry analysis at the different times.
RESULTS: RT-PCR analysis showed that HBV X gene could be expressed stably in HL-7702 cells. Both flow cytometry and TUNEL technology revealed that the apoptosis rates of HL-7702/HBx cells were much higher than those of HL-7702/ pcDNA3 and HL-7702 cells. Furthermore, the apoptotic phenomena and apoptotic body were observed in HL-7702/ HBx cells under electronic microscope, but not in HL-7702/ pcDNA3 and HL-7702 cells. In the experiment of transient transfection, RT-PCR reveals that X gene was expressed most at 72 h after transfection; and the apoptosis rate reached the highest at the same time. After that, the apoptosis rate was reduced with the decrease of the X gene expression.
CONCLUSION: HBV X gene and X protein can promote the apoptosis in hepatocyte. And there exist a quantity-effect relationship between the X gene expression and apoptosis rate in hepatocyte.
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Affiliation(s)
- Hong-Ying Chen
- Department of Gastroenterology, Union Hospital, Fujian Medical University, Fuzhou 350001, Fujian Province, China.
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Kirschberg O, Schüttler C, Repp R, Schaefer S. A multiplex-PCR to identify hepatitis B virus--enotypes A-F. J Clin Virol 2004; 29:39-43. [PMID: 14675868 DOI: 10.1016/s1386-6532(03)00084-2] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Eight genotypes (A-H) of hepatitis B virus (HBV) are known with variations in nucleotide sequences greater than 8%. Several recent publications found that the clinical course and outcome of antiviral therapy depended on the genotype of the infecting HBV strain. Large epidemiological studies will require the availability of a system which is rapid, reliable and can be performed on a large number of samples. We have developed a multiplex-PCR assay which uses genotype-specific primer pairs for HBV genotypes A-F. These primer pairs specifically amplified HBV DNA of the respective genotype, either in single or in multiplex-PCR. Sensitivity of the assay was in the range of 10(4) genome equivalents.
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Affiliation(s)
- Oliver Kirschberg
- Institut für Medizinische Virologie, Justus-Liebig-Universität, Giessen, Germany
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40
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Glebe D, Aliakbari M, Krass P, Knoop EV, Valerius KP, Gerlich WH. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus. J Virol 2003; 77:9511-21. [PMID: 12915565 PMCID: PMC187384 DOI: 10.1128/jvi.77.17.9511-9521.2003] [Citation(s) in RCA: 146] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2003] [Accepted: 06/03/2003] [Indexed: 12/17/2022] Open
Abstract
The susceptibility of the tree shrew Tupaia belangeri to human hepatitis B virus (HBV) has been demonstrated both in vivo and in vitro. In this study, we show that purified HBV infects primary T. belangeri hepatocyte cultures in a very specific manner, as detected by HBV covalently closed circular DNA, mRNA, HBV e antigen, and HBsAg production. A monoclonal antibody (MAb), MA18/7, directed against the pre-S1 domain of the large HBs protein, which has been shown to neutralize infectivity of HBV for primary human hepatocytes, also blocked infection of primary Tupaia hepatocytes. MAbs against the pre-S2 domain of HBs inhibited infection only partially, whereas an S MAb and polyvalent anti-HBs antibodies neutralized infection completely. Thus, both pre-S1 and S antigens are necessary for infection in the tupaia. Using subviral particles, >70% of primary Tupaia hepatocytes are capable of specific binding of pre-S1-rich HBsAg, showing localization in distinct membrane areas. The data show that the early steps of HBV infection in Tupaia hepatocyte cultures are comparable to those in the human system.
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Affiliation(s)
- Dieter Glebe
- Institute of Medical Virology. Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, 35392 Giessen, Germany.
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Lu YY, Chen TY, Cheng J, Liang YD, Wang L, Liu Y, Zhang J, Shao Q, Li K, Zhang LX. Cloning of a gene coding for novel mutant of asialoglycoprotein receptor 2 binding to hepatitis B virus X protein in hepatocytes. Shijie Huaren Xiaohua Zazhi 2003; 11:1126-1130. [DOI: 10.11569/wcjd.v11.i8.1126] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM The pathogenesis of HBV-induced malignant transformation is incompletely understood. The X protein of hepatitis B virus (HBxAg) is a multifunctional protein that can influence a variety of signal transduction pathways within the cell and is essential for establishing natural viral infection, it also has been implicated in the development of liver cancer associated with chronic infection. Further understanding of the interaction between HBxAg and proteins in hepatocytes is of great significance for the prevention of the development of hepatocellular carcinoma (HCC).
METHODS HBxAg bait plasmid was constructed by ligating the HBxAg gene with a yeast expression vector pGBKT7, then transformed into yeast AH109 (a type). The transformed yeast cells were amplified and mated with yeast cells Y187(α type) containing liver cDNA library plasmid pCAT2 in 2×YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selection twice. Plasmid of true positive blue colonies was extracted and analysed by DNA sequencing and blast in GenBank. After the complete sequence of the novel mutant of asialoglycoprotein receptor 2 (ASGPR2) was amplified from the mRNA of HepG2 cell by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pGADT7 vector, the recombined plasmid was translated by using reticulocyte lysate and analysed by immunoprecipitation technique in vitro together with HBxAg.
RESULTS Eighteen genes in forty-one positive colonies were obtained, one of them is a novel mutant of ASGPR2, which is 80 % homologous to natural ASGPR2. The complete sequence of the mutant was amplified from the mRNA of HepG2 cell by RT-PCR successfully. The interaction between HBx and ASGPR2 mutant was further confirmed by immunoprecipitation technique.
CONCLUSION Interaction between HBx and ASGPR2 mutant can be observed in both yeast cell and in vitro.
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Affiliation(s)
- Yin-Ying Lu
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Tian-Yan Chen
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Jun Cheng
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Yao-Dong Liang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Lin Wang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Yan Liu
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Jian Zhang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Qing Shao
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Ke Li
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Ling-Xia Zhang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
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42
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Shirakata Y, Koike K. Hepatitis B virus X protein induces cell death by causing loss of mitochondrial membrane potential. J Biol Chem 2003; 278:22071-8. [PMID: 12676947 DOI: 10.1074/jbc.m301606200] [Citation(s) in RCA: 130] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
The hepatitis B virus X protein (HBx) has been implicated in the carcinogenicity of this virus as a causative factor by means of its transactivation function in development of hepatocellular carcinoma. However, we and others have recently reported that HBx is located in mitochondria and causes subsequent cell death (Takada, S., Shirakata, Y., Kaneniwa, N., and Koike, K. (1999) Oncogene 18, 6965-6973; Rahmani, Z., Huh, K. W., Lasher, R., and Siddiqui, A. (2000) J. Virol. 74, 2840-2846). In this study, we, therefore, examined the mechanism of HBx-related cell death. Using enhanced green fluorescent protein (EGFP) fusion constructs of HBx, the region required for its mitochondrial localization was mapped to amino acids (aa) 68-117, which is essential for cell death but inactive for transactivation function. In vitro binding analysis supported the notion that the recombinant HBx associates with isolated mitochondria through the region of aa 68-117 without causing redistribution of cytochrome c and apoptosis-inducing factor (AIF). A cytochemical analysis revealed that mitochondrial membrane potential was decreased by HBx association with mitochondria, suggesting that HBx induces dysfunction of permeability transition pore (PTP) complex. Furthermore, PTP inhibitors, reactive oxygen species (ROS) scavengers and Bcl-xL, which are known to stabilize mitochondrial membrane potential, prevented HBx-induced cell death. Collectively, the present results suggest that location of HBx in mitochondria of hepatitis B virus-infected cells causes loss of mitochondrial membrane potential and subsequently induces mitochondria-dependent cell death.
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Affiliation(s)
- Yumiko Shirakata
- Department of Gene Research, The Cancer Institute (JFCR), Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455, Japan
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43
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Boya P, Roumier T, Andreau K, Gonzalez-Polo RA, Zamzami N, Castedo M, Kroemer G. Mitochondrion-targeted apoptosis regulators of viral origin. Biochem Biophys Res Commun 2003; 304:575-81. [PMID: 12729592 DOI: 10.1016/s0006-291x(03)00630-2] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
During coevolution with their hosts, viruses have "learned" to intercept or to activate the principal signal transducing pathways leading to cell death. A number of proteins from pathophysiologically relevant viruses are targeted to mitochondria and regulate (induce or inhibit) the apoptosis-associated permeabilization of mitochondrial membranes. Such proteins are encoded by human immunodeficiency virus 1, Kaposi's sarcoma-associated herpesvirus, human T-cell leukemia virus-1, hepatitis B virus, cytomegalovirus, and Epstein Barr virus, among others. Within mitochondria, such apoptosis regulators from viral origin can target distinct proteins from the Bcl-2 family and the permeability transition pore complex including the adenine nucleotide translocase, cyclophilin D, the voltage-dependent anion channel, and the peripheral benzodiazepine receptor. Thus, viral proteins can regulate apoptosis at the mitochondrial level by acting on a variety of different targets.
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Affiliation(s)
- Patricia Boya
- Centre National de la Recherche Scientifique, UMR 8125, Institut Gustave Roussy, Pavillon de Recherche 1, 39 rue Camille Desmoulins, F-94805 Villejuif, France
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44
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Kim KH, Seong BL. Pro-apoptotic function of HBV X protein is mediated by interaction with c-FLIP and enhancement of death-inducing signal. EMBO J 2003; 22:2104-16. [PMID: 12727877 PMCID: PMC156082 DOI: 10.1093/emboj/cdg210] [Citation(s) in RCA: 106] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Despite its implication in the progression of hepatitis B virus (HBV)-associated liver disease, the pro-apoptotic function of HBx protein remains poorly understood. We show that the expression of HBx leads to hyperactivation of caspase-8 and caspase-3 upon treatment with tumor necrosis factor-alpha (TNF-alpha) or anti-Fas antibody, and this activation is correlated with the sensitivity to apoptosis. We demonstrate cytoplasmic co-localization and direct interaction between HBx and the cellular FLICE inhibitory protein (c-FLIP), a key regulator of the death-inducing signaling complex (DISC). Deletion analysis shows that the death effector domain 1 (DED1) of c-FLIP is important for the observed interaction. Overexpression of c-FLIP rescued the cells from HBx-mediated apoptosis, with both the full-length HBV genome and HBx expression vectors. Moreover, c-FLIP and caspase-8 inhibitor considerably protected cells from HBx-mediated apoptosis. These data suggest that HBx abrogates the apoptosis-inhibitory function of c-FLIP and renders the cell hypersensitive towards the TNF-alpha apoptotic signal even below threshold concentration. This provides a novel mechanism for deregulation of hepatic cell growth in HBV patients and a new target for intervention in HBV-associated liver cancer and disease.
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Affiliation(s)
- Kyun-Hwan Kim
- Department of Biotechnology, College of Engineering and Bioproducts Research Center, Yonsei University, Seoul 120-749, Korea
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45
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Biermer M, Puro R, Schneider RJ. Tumor necrosis factor alpha inhibition of hepatitis B virus replication involves disruption of capsid Integrity through activation of NF-kappaB. J Virol 2003; 77:4033-42. [PMID: 12634363 PMCID: PMC150632 DOI: 10.1128/jvi.77.7.4033-4042.2003] [Citation(s) in RCA: 144] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Chronic infection by hepatitis B virus results from an inability to clear the virus, which is associated with liver disease and liver cancer. Tumor necrosis factor alpha (TNF-alpha) is associated with noncytopathic clearance of hepatitis B virus in animal models. Here we demonstrate that the nuclear factor kappaB (NF-kappaB) signaling pathway is a central mediator of inhibition of hepatitis B virus by TNF-alpha and we describe the molecular mechanism. TNF-alpha is shown to suppress hepatitis B virus DNA replication without cell killing by disrupting the formation or stability of cytoplasmic viral capsids through a pathway requiring the NF-kappaB-activating inhibitor of kappaB kinase complex IKK-alpha/beta and active transcription factor NF-kappaB. Hepatitis B virus replication could also be inhibited and viral capsid formation could be disrupted in the absence of TNF-alpha solely by overexpression of IKK-alpha/beta or strong activation of NF-kappaB. In contrast, inhibition of NF-kappaB signaling stimulated viral replication, demonstrating that HBV replication is both positively and negatively regulated by the level of activity of the NF-kappaB pathway. Studies are presented that exclude the possibility that HBV inhibition by NF-kappaB is carried out by secondary production of gamma interferon or alpha/beta interferon. These results identify a novel mechanism for noncytopathic suppression of hepatitis B virus replication that is mediated by the NF-kappaB signaling pathway and activated by TNF-alpha.
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Affiliation(s)
- Michael Biermer
- Department of Microbiology, New York University School of Medicine, New York, New York 10016, USA
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46
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Schuster R, Hildt E, Chang SF, Terradillos O, Pollicino T, Lanford R, Gerlich WH, Will H, Schaefer S. Conserved transactivating and pro-apoptotic functions of hepadnaviral X protein in ortho- and avihepadnaviruses. Oncogene 2002; 21:6606-13. [PMID: 12242658 DOI: 10.1038/sj.onc.1205916] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2001] [Revised: 07/01/2002] [Accepted: 07/25/2002] [Indexed: 01/12/2023]
Abstract
Two established activities of the multifunctional human hepatitis B virus X-protein are its transactivating and pro-apoptotic potential. We analysed whether X-proteins from other orthohepadnaviruses and the newly discovered avihepadnaviral X-proteins have similar functions as HBx. Previously, we have shown that HBx suppresses oncogenic transformation of primary rat embryo fibroblasts (REF) by induction of apoptosis. Using this system, we found that the wildtype X-proteins of woodchuck, ground squirrel, arctic squirrel and woolly monkey hepatitis B virus exhibit similar levels of pro-apoptotic activity as HBx, whereas mutants with carboxyterminal deletions were severely impaired in this activity. A strong correlation between the pro-apoptotic and transactivating abilities of the mammalian X-proteins was found. The newly discovered avihepadnaviral X-like proteins showed similar and Raf-MAPK pathway-dependent transactivating abilities and induced apoptosis in the REF-assay. Our data indicate that the transactivating and pro-apoptotic activities reside in the carboxyterminal half of orthohepadnaviral X and are conserved in avihepadnaviral X-proteins.
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47
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Yun C, Um HR, Jin YH, Wang JH, Lee MO, Park S, Lee JH, Cho H. NF-kappaB activation by hepatitis B virus X (HBx) protein shifts the cellular fate toward survival. Cancer Lett 2002; 184:97-104. [PMID: 12104053 DOI: 10.1016/s0304-3835(02)00187-8] [Citation(s) in RCA: 67] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
In this paper, we examined the cellular effect of hepatitits B virus X (HBx) in ChangX-34 cells, inducible HBx-expressing cells. High expression of HBx protein in ChangX-34 cells resulted in approximately three-fold increase in DNA synthesis and did not show apoptotic changes. Expression of HBx in these cells was accompanied by the NF-kappaB-mediated transcription. Interestingly, inhibition of NF-kappaB activity either by treatment with sulfasalazine, a specific inhibitor of NF-kappaB, or by expressing IkappaBalpha super-repressor significantly increased cell death in ChangX-34 cells but had no influence on parental Chang cells. Thus, the activation of NF-kappaB in HBx-expressing cells may play a critical role in shifting the balance toward cell survival.
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Affiliation(s)
- Chawon Yun
- Department of Biochemistry, Ajou University School of Medicine, Suwon 442-749, South Korea
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48
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Abstract
Hepatocellular carcinoma (HCC) is one of the most common reasons for malignancy-related death in Africa and Asia and is still recognised as the leading cancer in men in Taiwan. Despite enthusiastic efforts in early diagnosis, aggressive surgical treatment and application of additional nonoperative modalities, its prognosis is still dismal. This emphasises the necessity to develop new measures and strategies for its prevention. Inducible cyclooxygenease 2 (COX-2) is an immediate-early (IE) response gene and extensive studies conducted over the past few years have recognised its overexpression in several carcinomas and thus its implication in carcinogenesis. Recent studies have suggested that overexpression of COX-2 might be one of the leading factors in hepatic carcinogenesis. COX-2 can induce angiogenesis via vascular endothelial growth factor (VEGF) and prostaglandin production and can also inhibit apoptosis by inducing the antiapoptotic factor Bcl-2 as well as activating antiapoptotic signalling through Akt/PKB. Therefore, the use of selective inhibitors for the downregulation of COX-2 activity might be a target for preventing hepatic carcinoma development.
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Affiliation(s)
- Mohammad A Rahman
- Second Department of Surgery, Shimane Medical University, Izumo 693-8501, Japan.
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49
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Tralhao JG, Roudier J, Morosan S, Giannini C, Tu H, Goulenok C, Carnot F, Zavala F, Joulin V, Kremsdorf D, Bréchot C. Paracrine in vivo inhibitory effects of hepatitis B virus X protein (HBx) on liver cell proliferation: an alternative mechanism of HBx-related pathogenesis. Proc Natl Acad Sci U S A 2002; 99:6991-6996. [PMID: 12011457 PMCID: PMC124516 DOI: 10.1073/pnas.092657699] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2001] [Indexed: 12/16/2022] Open
Abstract
The role of the hepatitis B virus X protein (HBx) in the pathogenesis of hepatitis B virus (HBV) infection remains unclear. HBx exhibits pleiotropic biological effects, whose in vivo relevance is a matter for debate. In the present report, we have used a combination of HBx-expressing transgenic mice and liver cell transplantation to investigate the in vivo impact of HBx expression on liver cell proliferation and viability in a regenerative context. We show that moderate HBx expression inhibits liver regeneration after partial hepatectomy in HBx-expressing transgenic mice. We also demonstrate that the transplantation of HBx-expressing liver cells, isolated from HBx transgenic mice, is sufficient to inhibit overall recipient liver regeneration after partial hepatectomy. Moreover, the injection of serum samples drawn from HBx-expressing transgenic mice mimicked the inhibitory effect of HBx on liver regeneration. Finally, the incubation of primary rat hepatocytes with the supernatant of HBx-expressing liver cells inhibits cellular DNA synthesis. Taken together, our results demonstrate a paracrine inhibitory effect of HBx on liver cell proliferation and lead us to propose HBV as one of the few viruses implicated in human cancer which act, at least in part, through paracrine biological pathways.
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Affiliation(s)
- J Guilherme Tralhao
- Institut Pasteur-Institut National de la Santé et de la Recherche Médicale Unit 370, Necker Institute, 75015 Paris, France
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50
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Terradillos O, de La Coste A, Pollicino T, Neuveut C, Sitterlin D, Lecoeur H, Gougeon ML, Kahn A, Buendia MA. The hepatitis B virus X protein abrogates Bcl-2-mediated protection against Fas apoptosis in the liver. Oncogene 2002; 21:377-86. [PMID: 11821950 DOI: 10.1038/sj.onc.1205110] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2001] [Revised: 10/08/2001] [Accepted: 10/29/2001] [Indexed: 02/06/2023]
Abstract
The role of the hepatitis B virus protein HBx in liver cell proliferation and apoptosis remains controversial. Using a transgenic mouse model, we have recently shown that HBx stimulates the apoptotic turnover of hepatocytes, independently of p53. In this paper, we tested whether the proapoptotic function of HBx can interfere with Bcl-2 during hepatic apoptosis in vivo. HBx transgenic mice were crossed with PK-hBcl-2 mice that are protected against Fas killing by constitutive overexpression of Bcl-2 in hepatocytes. In a lethal challenge with Fas antibodies, HBx expressed at low levels restored sensitivity to Fas-mediated apoptosis and fulminant hepatic failure in mice overexpressing Bcl-2. Furthermore, cytochrome c release from mitochondria and caspase 3 activation were restored to normal levels in HBx/Bcl-2 mice during transduction of the Fas signal. Thus, the proapoptotic activity of HBx overcomes or bypasses the inhibitory effect of Bcl-2 against Fas cytotoxicity. This effect was not apparently mediated through downregulation of the PK-hBcl-2 transgene or via delocalization of the Bcl-2 protein, and a direct interaction of HBx with Bcl-2, Bcl-X(L) or Bax could not be evidenced in yeast two-hybrid assays. We further show that apoptosis induced by ectopic expression of HBx is associated with mitochondrial membrane alterations and caspase 3 activation. Our data indicate that the dominant function of HBx upon Bcl-2-regulated control of apoptosis might play an important role in the pathogenesis of chronic hepatitis B.
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Affiliation(s)
- Olivier Terradillos
- Unité de Recombinaison et Expression Génétique, Inserm U163, Institut Pasteur, Paris, France
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