Epidemiological studies indicate that chronic infection with Hepatitis B Virus (HBV) is the leading cause for the development of hepatocellular carcinoma (HCC) [1]. According to the most recent data from Globocan 2008, which estimates the incidence, prevalence, and mortality of common cancers in the world, HCC is the fifth most common cancer in men and seventh most common in women [2]. Worldwide there were an estimated 694,000 deaths due to HCC in 2008 [2]. More than 50% of HCC cases worldwide and 70%–80% of HCC cases in HBV endemic areas are due to chronic HBV infection. Despite the availability of an HBV vaccine, approximately 350 million people are chronically infected with HBV worldwide, and HBV infection-related liver diseases remain a major global health problem [1].

HBV belongs to the Hepadnaviridae family of viruses. HBV has a highly compact genome of only 3.2 kilobases (kB) in length; this genome contains four overlapping open reading frames (ORFs) that encode the viral core protein (capsid), surface proteins (envelope), reverse transcriptase, and X protein (HBx). The viral genome is a partially double stranded, relaxed circular DNA that replicates by reverse transcription of an RNA intermediate. Hepatocytes are the predominant cells infected by HBV, and HBV host range and cell tropism are controlled by interactions between the virus and a cell surface receptor as well as hepatocyte-specific intracellular factors [3,4,5,6]. The cell surface receptor of HBV is not known, and the mechanism by which HBV enters hepatocytes is not clear. Although a recent report suggests that HBV enters hepatocytes through clathrin dependent endocytosis [7]. Following entry into hepatocytes, the HBV genome is delivered to the nucleus where the host-cell DNA repair machinery repairs the partially double-stranded DNA of the viral genome to form covalently closed circular DNA (cccDNA) (Figure 1). cccDNA is the template for transcription of viral mRNAs including the pre‑genomic RNA (pgRNA). Viral transcripts are then exported out of the nucleus into the cytoplasm and translated to form various viral proteins. pgRNA is packaged into viral capsids and is reverse transcribed by the virally encoded reverse transcriptase, which remains associated with the pgRNA, to generate the first DNA strand of the viral genome. The second complementary DNA strand of the viral genome is then completed to varying lengths, which gives rise to the partially double stranded DNA genome. Viral surface proteins envelop the capsids at the endoplasmic reticulum, and the packaged viral particles are secreted from cells using the secretory machinery of the host [8]. The envelope protein of HBV co-localizes with the proteins of multivesicular bodies in human hepatoma cells, and host multivesicular body functions are required for the release of enveloped HBV virions [9]. Some of the newly replicated genome in core particles is not secreted from cells but is instead delivered back to the nucleus to maintain a stable pool of nuclear cccDNA [8].

Although the association of chronic HBV infection with the development of HCC is well established, the exact molecular mechanisms or events that link HBV infection to HCC development are not completely defined. Three factors have been proposed to influence HCC development in a chronically HBV infected individual. These factors are: integration of viral DNA into the genome of the host cell, chronic inflammation due to the immune response of the host to the HBV infection, and HBV encoded proteins, such as HBx, that modulate cellular signal transduction pathways [10,11,12,13,14].

Integration of HBV DNA into the host cell genome has been observed in hepatocytes of chronically HBV-infected patients [15,16,17,18]. The integrated HBV DNA has been detected around the telomerase gene [19], cancer susceptibility regions, fragile sites [10,20], and the cyclin A gene [21]. Although these observations might suggest that there are preferred sites for HBV integration into the host genome, integration events were also reported to be random [22], and it remains unclear whether putative preferred integration sites reflect actual preferential integration or selective pressure for random integration events that are ultimately advantageous for cell survival and transformation. Some studies have suggested that the HBV HBx gene, and more specifically, truncated forms of the HBx gene, is preferentially integrated into the host cell DNA [23,24]. It is important to note, however, that studies have shown that an aberrant HBV replication intermediate, a double-stranded linear form of the viral genome, is a preferred form of the HBV genome that is integrated as compared to the circularized form of the genome [25]; this linear form of the viral genome has at its end the HBx gene. Therefore, the observed high frequency of integrated forms of the HBx gene may simply reflect preferential integration of a double-stranded linear form of the viral genome, or a double-stranded linear form of the HBV genome in which the ends of the linear HBV genome have been exposed to exonucleases prior to integration, rather than a preferential integration of the HBx gene. Overall, the relationship between integration of HBV DNA into the host genome and HCC development needs further investigation. For a description of the potential link between integration of HBV DNA into the host genome and the development of HCC, readers are referred to other more comprehensive reviews of this topic [10,11].

The second factor that is thought to have an important role in the development of HBV-associated HCC is the host immune response against the HBV infection, which is crucial for clearing the viral infection [4]. In chronically HBV infected individuals, immune mediated clearance of HBV-infected hepatocytes stimulates both clearance of HBV-infected hepatocytes and liver regeneration. During these cycles of hepatocyte clearance and regeneration, hepatocytes can accumulate mutations that ultimately contribute to transformation processes [14,26]. In addition, the immune response to an HBV infection causes infiltration of immune cells, release of inflammatory cytokines that can alter cell signaling pathways, and generation of reactive oxygen species (ROS) that can create a mutagenic environment leading to hepatocyte transformation [4,14]. Inflammation has been linked to many types of cancers, and changes in the microenvironment due to inflammation can influence angiogenesis and tumor cell metastasis [27]. Multiple pathways associated with inflammation, such as activation of the transcription factors Nuclear Factor kappa B (NF-κB) and Signal Transducer and Activator of Transcription 3 (STAT-3), cytokine production, and alterations of cellular signaling pathways can result in the development of HCC and are associated with an HBV infection [14,28]. Although the role of inflammation in the development of HBV-associated HCC is well established, a recent report of HCC development in HBV-transgenic mice suggests that HBV itself can be oncogenic in the absence of an inflammatory response [29]. For a comprehensive description of the role of the host immune response in HBV infection and HCC development readers are referred to recently published reviews [12,13].

The third factor that is thought to play a role in the development of HBV-associated HCC, and the subject of this review, is activities of the HBx protein in HBV-infected cells [14,30,31,32,33]. HBx is the major regulatory protein encoded by the HBV genome, and the results of numerous studies strongly suggest that HBx is involved in the development of HBV-associated HCC [14,34]. Although both mammalian and avian hepadnaviruses can establish chronic infections in their hosts, only chronic infections with the mammalian hepadnaviruses are associated with the development of liver cancer, and only the mammalian hepadnaviruses encode an X protein [6]. Avian hepadnaviruses either completely lack an X protein or encode a highly divergent form of this protein [35]. Studies conducted in cell culture and HBx-transgenic mice have also suggested a cofactor role of HBx in the development of HBV associated HCC (discussed in the next section). One controversial aspect of HBx biology that may be involved in the development of HBV-associated HCC is whether HBx modulates apoptotic pathways. HBx has been shown to stimulate, inhibit, or have no impact on apoptotic pathways depending on the conditions of a particular study [36,37,38,39,40,41]. In this review, we provide a brief overview of HBx biology, a review of the reported impact of HBx expression on apoptotic pathways, and a discussion of the potential role of HBx modulation of apoptosis in the development of HCC. Alterations of normal apoptotic processes have been linked to the development of HCC [42], suggesting that an understanding of how HBx impacts apoptotic pathways in hepatocytes may help identify molecular mechanisms that influence the development of HBV-associated HCC and potential contributions of HBx in this process.

The smallest open reading frame of mammalian hepadnavirus genomes encodes the 17 kilodaltons (kDa), multi-functional X protein. HBx, which is encoded by the human hepadnavirus HBV, is predominantly nuclear at low expression levels and mostly cytoplasmic at high expression levels; a fraction of cytosolic HBx co-localizes with mitochondria [36,43,44,45,46,47,48,49]. In HepG2 cells, a human hepatoblastoma cell line, and cultured primary rat hepatocytes, mitochondrial HBx is localized in the outer mitochondrial membrane, which is consistent with the reported interaction of HBx with the voltage-dependent anion channel (VDAC), a channel that spans the outer mitochondrial membrane and is thought to be a component of the mitochondrial permeability transition pore (MPTP) [43,44,50].

Although originally controversial due to the absence of any effect of HBx on HBV replication in human hepatoma Huh7 cells, there is now considerable evidence to support the notion that HBx is required for HBV replication [51]. HBx was shown to play a crucial role in HBV replication in cultured primary rat hepatocytes and HepG2 cells, and a recent report even suggested that HBx stimulates HBV replication in Huh7 cells [38,52,53,54,55,56]. The reason for the different outcomes in Huh7 cells is not known. While not absolutely required for HBV replication, HBx was also shown to enhance HBV replication in an HBV-transgenic mouse model [57]. In a more recent study in which a wild-type or HBx-deficient HBV genome was hydrodynamically injected into mice, the absence of HBx caused a 75% reduction in HBV replication in mouse hepatocytes and a 99% reduction in the level of circulating HBV [58]. In another study, a human hepatocyte chimeric mouse model in which mice hepatocytes are replaced with human hepatocytes was used to study the effect of HBx on HBV replication. The results of this study demonstrated that wild-type HBV infection resulted in measurable viremia after 2–7 weeks, whereas HBx-deficient HBV infection did not result in a measurable viremia up to 16 weeks after injection of the HBx-deficient HBV. However, HBV replication in HBx-deficient HBV-infected human hepatocytes in these chimeric mice was rescued by hydrodynamic tail vein injection of an HBx expression plasmid [59]. Finally, studies conducted by directly infecting cultured primary human hepatocytes with wild-type HBV or an HBx-deficient HBV also demonstrated the requirement for HBx in HBV replication [60]. Collectively, these studies provide evidence that HBx strongly stimulates and is likely required for HBV replication in normal hepatocytes.

HBx can regulate cellular transcription, proliferation, DNA repair, apoptosis, and protein-degradation pathways, and these HBx activities have been invoked as potential mechanisms that link HBx expression and HBV replication to the development of HCC (Figure 2) [38,61,62,63,64,65,66]. HBx activities such as modulation of calcium signaling and regulation of the cell cycle have been shown to be important for HBV replication [55,65,67,68,69,70]. Whether HBx directly contributes to the development of HBV-associated HCC or functions as a co-factor in HCC development continues to be debated. In some HBx transgenic mouse models, HBx that was expressed in the absence of other HBV proteins directly facilitated the development of HCC [30,71]. In contrast, in other HBx-transgenic mouse models, HBx expression did not directly induce tumor formation but instead sensitized hepatocytes to carcinogen-induced HCC, suggesting a cofactor role of HBx in HCC development [72,73]. In a study conducted in HBV transgenic mice, HBV by itself could not induce HCC development but sensitized hepatocytes to diethylnitosamine (DEN) induced carcinogenesis [74]. In this model HBx was not essential for promoting DEN induced carcinogenesis but did enhance the efficiency of DEN induced carcinogenesis, possibly due to the effects of HBx on cellular signaling pathways or impairment of DNA repair [74]. More recently, the same group that earlier demonstrated that HBV transgenic mice were susceptible to DEN-induced carcinogenesis, but otherwise did not develop HCC up to 1 year of age, showed that these HBV transgenic mice when allowed to age for 2 years develop HCC in the absence of any carcinogenic treatment. Significantly, inflammation was not observed in the livers of the HBV-transgenic mice that developed HCC, suggesting that HBV on its own is carcinogenic [29]. Because a comparison was not made to HCC development in HBx-deficient HBV transgenic mice over the same time course, whether HBx directly influenced HCC development in the liver of these HBV-transgenic mice cannot be evaluated [29]. HBx has also been shown to decrease telomerase activity in HBx transgenic mice after a partial hepatectomy, suggesting that in the regenerating liver, HBx expression might lead to shortening of telomeres, which can cause chromosomal instability and contribute to malignant transformation [75]. In another study, HBx/c-myc bi-transgenic mice were created by crossing transgenic mice that expressed HBx in hepatocytes with transgenic mice that have liver-specific c-myc expression driven by woodchuck hepatitis virus (WHV). While HBx was not directly oncogenic in the original HBx transgenic mice, the HBx/c-myc bi-transgenic mice developed liver tumors, and the tumors in bitransgenic mice developed more rapidly as compared to WHV/c-myc transgenic mice, suggesting a cofactor role of HBx in c-myc induced tumor development [76]. Although the studies in the various HBx-transgenic mouse models have identified what appear to be mouse-strain specific responses to HBx expression, they none-the-less suggest that HBx can participate in hepatocyte transformation [37,71,73,77,78,79]. Overall, the results of most studies that have analyzed the impact of HBx expression on cellular signaling pathways that may contribute to hepatocyte transformation support a co-factor role for HBx in the development of HBV-associated HCC. A co-factor role for HBx in HBV-associated HCC is also more consistent with the biology of an HBV infection; although the entire hepatocyte population of the liver can be infected during a chronic HBV infection, transformation only occurs in a small number of hepatocytes and usually requires decades of a chronic HBV infection [3].

It is possible that multiple HBx activities contribute to the development of HBV-associated HCC, and many of the signaling pathways or cellular proteins that are regulated by HBx provide plausible mechanisms that could link HBx expression to the development of HBV-associated HCC [14,34]. While inflammation-mediated destruction of HBV-infected hepatocytes and subsequent liver regeneration contribute to the development of HBV-associated HCC, the results of studies in mice, established cell lines, and cultured primary hepatocytes suggest that activities associated with expression of HBx can regulate HBV replication, alter hepatocyte physiology, and modulate cell signaling pathways that may impact the development of HBV-associated HCC [14,34]. A critical evaluation of published studies supports the notion that HBx activities that influence HCC development are subtle. In fact, it is currently easier to study the role of HBx during HBV replication in animal models and tissue culture models than to assess the role of HBx in HCC development, which can take up to 30 years in a chronically HBV-infected individual [80]. During this time course of a chronic HBV infection, there can be fluctuations in the number of hepatocytes that are infected with HBV, the turnover of hepatocytes, the level of HBV replication, the expression of HBV proteins, and the immune response to the HBV infection [8,81,82], making it very difficult to directly assess the role of HBx in the development of HBV-associated HCC. With this as a consideration, one HBx activity that could directly impact the development of HBV-associated HCC is HBx modulation of apoptotic pathways [14,34]. De-regulation of normal hepatocyte apoptotic pathways is a well‑established mechanism that contributes to HCC development and progression [83,84]. Although discrepant observations have been published, there is considerable evidence that HBx can regulate apoptotic pathways, and this review will now focus on this HBx activity and its potential contribution to the development of HBV-associated HCC.

Apoptosis, or programmed cell death, is a highly regulated process that has a vital role in organ development and clearance of diseased or damaged cells [85]. Apoptosis can be initiated by extrinsic (originating outside a cell) or intrinsic (originating inside a cell) signaling pathways [86]. In the extrinsic pathway, binding of an extracellular ligand such as Fas to its receptor on the cell surface transmits a signal that activates an intracellular chain of events that cause cleavage and activation of procaspase-8 to form caspase-8. Caspases are cysteine-aspartate proteases that are essential for apoptosis [87]. Caspases can be initiator caspases such as caspase 2, 8, 9, and 10, which cleave the inactive form of procaspases, or they can be effector/executioner caspases such as caspases 3, 6, and 7, which cleave other protein substrates in cells. Caspase-8 is an initiator caspase that is activated in the extrinsic pathway of apoptosis and cleaves downstream caspases to eventually activate the executioner caspase, caspase-3 [87]. The intrinsic pathway is initiated by the release of cytochrome c from mitochondria along with other apoptosis-inducing factors such as second mitochondria-derived activator of apoptosis (Smac)/direct IAP binding protein with low pI (DIABLO) [86]. Cytochrome c forms an apoptosome complex with procaspase-9 and other apoptosis-regulatory proteins. Procaspase‑9 is cleaved to form caspase-9, which cleaves downstream caspases and activates caspase‑3. Caspase-3 cleaves various substrates such as Poly ADP-ribose polymerase (PARP), that ultimately causes the morphological and biochemical changes typically observed in apoptotic cells [85].

Depending upon the mechanism of apoptosis, cells can be classified as type I cells that require only caspase 8 mediated activation of executioner caspases or type II cells that require activation of caspase 8 as well as mitochondrial outer membrane permeabilization (MOMP) to release mitochondria-localized proteins that activate apoptotic pathways [86]. The crosstalk between extrinsic and intrinsic apoptotic pathways can be regulated by caspase 8-mediated cleavage and activation of BCL-2 homology 3 (BH3)-interacting domain death agonist (BID) [88,89]. BID in turn causes MOMP, which initiates the intrinsic pathway of apoptosis. The requirement for MOMP discriminates between type I and type II cells in death receptor mediated apoptosis [86]. Hepatocytes are type II cells and require the activation of BID followed by MOMP for complete activation of apoptosis [86]. This is demonstrated by the fact that anti-Fas ligand antibodies injected into mice cause hepatocellular apoptosis, but mice that are deficient in BID are protected when injected with the anti-Fas antibody and have no liver injury or activation of caspases 3 or 7, even though the initiator caspase 8 is activated [90]. Readers are referred to other comprehensive reviews for a detailed explanation of the role of MOMP in apoptosis [86,91,92].

A large number of studies have assessed the impact of HBx expression on the regulation of apoptotic pathways, but the results of these studies have varied. HBx has been shown to induce [36,37,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107], inhibit [38,39,108,109,110,111,112,113] or have no effect on apoptosis [40,41,114] in multiple cellular contexts (Figure 3). In addition, some studies have demonstrated that HBx does not directly induce apoptosis in certain cell types, but instead sensitizes these cells to pro-apoptotic stimuli [97,99,107]. It is likely that the seemingly discrepant apoptotic activities of HBx reflect differences in cell types used for these assays, whether studies were conducted in cells with transiently transfected versus constitutively expressed HBx, which may have facilitated cellular adaptation to HBx activities, and whether transgenic mouse hepatocytes expressed HBx at the time of analysis or had adapted to the continued expression of HBx during liver development. Some of the studies analyzing the effects of HBx on cellular apoptosis have also been conducted in non-hepatic cell lines, and since HBV does not infect these cells the relevance of the conclusions from such studies is uncertain. The potential influence of cellular context for HBx activities has been clearly demonstrated in studies using AML12 cells, a hepatocyte cell line derived from TGF-α transgenic mice [115]. During these studies in AML12 cells, stable HBx-expressing cell lines were generated in which HBx expression was controlled by a tetracycline responsive element. Two of these new AML12 derivative cell lines, 3pX-1 and 4pX-1, showed different extents of hepatocyte differentiation. Surprisingly, HBx sensitized de‑differentiated 4pX-1 cells, but not differentiated 3pX-1 cells to the activation of apoptotic pathways [99]. These observations, combined with reports that HBx can have different effects on apoptotic pathways in various experimental systems, supports the notion that the exact consequence of HBx expression on modulation of apoptotic pathways is likely influenced by the characteristics of the cells used for a particular study and demonstrate the importance of conducting experiments in systems that closely mimic physiologically relevant conditions. In the following sections, we will separately summarize studies that have demonstrated a pro-apoptotic effect of HBx and studies that have demonstrated an anti-apoptotic effect of HBx.

The results of the numerous studies highlighted in this review provide compelling evidence that HBx can regulate cellular apoptotic pathways. The use of different cellular and transgenic mouse model systems, different levels of HBx expression, the presence or absence of growth factors, or whether the study was conducted in the context of HBV replication have sometimes generated contradictory observations. Collectively, the results of various studies suggest that HBx is capable of eliciting either an anti- or pro-apoptotic response and that this HBx activity is cell-context dependent. The cell-context specific effect of HBx presents the interesting possibility that the ability of HBx to induce or inhibit apoptosis may change during the course of an HBV infection as the liver is undergoing regeneration or as hepatocytes are undergoing transformation or responding to immune-cell-secreted cytokines, such as TNFα [163,164]. It is also possible that HBx has different effects in different microenvironments of the liver depending on the expression of key transcription factors, such as NF-κB, in that area. It is known that certain transcription factors are present at varying levels in different hepatic zones, and these local signals could affect HBx activities in different areas of the liver [165,166,167].

In studies in cultured primary rat hepatocytes, HBx was both pro- and anti-apoptotic, depending on the status of NF-κB activation. HBx activation of NF-κB prevented hepatocyte apoptosis, whereas in the absence of NF-κB activity, the ability of HBx to modulate the MPTP stimulated apoptosis [38]. While NF-κB inactivation inhibits carcinogenesis in some cell types [168], it can promote carcinogenesis in hepatocytes [126], and both inhibition and stimulation of apoptosis have been implicated in the development of HCC. Studies conducted in multidrug resistance 2 (Mdr2)-knockout mice demonstrated that NF-κB-dependent inhibition of apoptosis can promote liver tumor formation, and inhibition of NF-κB activity in these mice diminished tumor formation [169]. Conversely, repeated cycles of apoptosis and compensatory regeneration could eventually participate in the selection of hepatocytes that are resistant to pro-apoptotic signals and contribute to the development of HCC. For example, mice lacking IKKβ expression undergo more apoptosis and develop higher numbers of liver tumors than wild-type mice [126]. The results of one study conducted in HBx‑transgenic mouse demonstrated a delayed HCC development due to increased DNA synthesis and compensatory apoptosis [37]. HBx activation of NF-κB and inhibition of apoptosis could provide an environment that favors cell survival and tumor development. HBx-induced apoptosis and commensurate liver regeneration, which could occur in the context of diminished NF-kB activity in certain hepatic zones or as cells respond to various cytokines, could contribute to the eventual selection of hepatocytes that are resistant to pro-apoptotic signals, also resulting in the development of HCC. Cumulatively, the results of various studies suggest that during a chronic HBV infection of the liver, HBx could either induce or inhibit apoptosis, depending on the status of NF-κB activity and that both of these HBx effects could contribute to the development of HBV-associated HCC.

In summary, although it appears that HBx can regulate apoptosis, it is likely that in different cellular contexts the regulation of cellular signaling pathways by HBx is different. The development of HCC in a chronically HBV infected individual is a long process, and during this process the dominant cellular signaling pathways, microenvironment of the liver, and factors such as ROS might play an important role in defining the regulation of apoptosis by HBx. Different model systems used to study HBx regulation of apoptosis might provide clues regarding HBx apoptotic activities in hepatocytes as they progress through various stages of HCC development. The development of more human-relevant model systems, such as three-dimensional (3D) mini-liver systems composed of human liver cells will be critical in furthering our understanding of the role of HBV and apoptosis in the development of liver cancer [170,171,172] Although studies in primary hepatocytes support the notion that HBx is anti‑apoptotic and that this HBx activity is NF-κB dependent, seemingly discrepant observations in various system may be providing essential insights into cell-context specific activities of HBx that could also influence HBV replication, apoptosis, and HCC development. It is clear that additional studies are required to define mechanisms that underlie HBx regulation of apoptosis during a chronic HBV infection, how the liver environment influences this HBx activity, and how this HBx activity might change as hepatocytes undergo transformational processes. Importantly, a clear understanding of HBx regulation of apoptotic pathways during an HBV infection will help define mechanisms that regulate HBV replication and influence HCC development and may identify novel therapeutic strategies for preventing the development of HBV-associated HCC.