Expression of junB is markedly stimulated by glycyrrhizin in a human hepatoma cell line.

Effective therapeutic approaches for liver cancer are expected to be the prevention of chronic inflammation, progression of chronic liver injury to liver cancer and/or tumor cell growth by activating various oncogenes. Activation of the c-jun oncogene occurs in many cases at the early stage of transformation of chronic hepatitis into liver cancer. Accordingly, inhibition of c-jun gene function is thought to be important for the therapy of liver cancer. Although the junB gene has been identified as a c-jun-related gene, it acts as a tumor suppressor gene through competitive binding of JUNB with c-JUN. Therefore, alteration in junB gene expression in chronic hepatitis or liver cancer is an interesting target for the development of both therapeutic treatment and medicines. Monoammonium glycyrrhizinate (MAG) is used for the treatment of viral hepatitis or the prevention of chronic liver diseases. However, the mechanism by which MAG is involved in the suppression of oncogene function has not yet been characterized. In the present study, we first found that MAG highly stimulated JUNB expression in a human hepatoma cell line, HepG2. We examined the mechanism by which MAG increases junB gene expression by considering the previously published effects of MAG on the onset or development of chronic hepatitis or liver injury. The present data suggest that marked activation of junB expression leads to a pivotal role for MAG in multiple medical applications.

Hepatitis B virus X gene is implicated in liver carcinogenesis.

Hepatitis B virus (HBV) is a small hepatotropic and highly species-specific enveloped DNA virus. The carcinogenicity of this virus has become focused on the X gene and its coded X protein. The X protein itself is unable to bind to DNA directly, but works as a potent transcriptional activator through multiple cis-acting elements and mediates several signal transduction cascades. Two regions of the X protein, aa.61-69 and aa.105-140, are found essential for the viral replication and expression as well. These functions interacting with transcription factors and signaling cascades are acting cooperatively to cause cell proliferation. Furthermore, the association of X protein with mitochondria causes loss of the mitochondrial membrane potential and subsequently causes cell death, the function of which is attributed to the aa.68-104 region of X protein. As a result, the X protein has two independent proliferative and cell death-promoting activities. Liver cancer has been shown to result from a series of mutations in specific oncogenes and tumor suppressor genes. In a recent study, X protein stimulates ROS generation in the mitochondria due to collapse of the membrane potential and increases the mutation frequency, that evokes malignant transformation. Inflammation as a result of HBV infection is concerned to cause DNA damage. In the past 10years, the possibility that several viral proteins directly engaged in the DNA damage has increased to some extent. From an evolutionary viewpoint, it is noteworthy that several arrangement proteins have been found in viruses. Thus, there is some clue that a small amount of X protein acts as an arrangement protein for HBV replication dependent upon cellular DNA damage due to generated ROS as an amplified signal.

Cell density-dependent regulation of tumor necrosis factor alpha gene expression in a human hepatoma cell line.

Human tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine expressed in many cell types. Although the TNFalpha gene expression in human hepatocytes has been detected previously, its regulation is not well understood yet. In this study, we demonstrated that TNFalpha gene expression in human hepatoma cell line, huH2-2, was activated as a function of cell density. TNFalpha mRNA expression was low in the low-density culture, while significantly high expression was detected in the high-density culture. Moreover, stability of TNFalpha mRNA was not changed by cell density, eliminating a possibility of post-transcriptional regulation. Antibody neutralization against human TNFalpha had no significant effect on the TNFalpha mRNA expression. A cellular factor for the TNFalpha gene expression is suggested to be accumulated in the high-density cells. Data indicate that the level of TNFalpha gene transcription is elevated by a cellular factor in a cell density-dependent manner without influencing the TNFalpha secretion under the present cell-culture conditions used.

Nucleotide sequence variation is frequent in the mitochondrial DNA displacement loop region of individual human tumor cells.

The mitochondrial DNA (mtDNA) displacement loop (D-loop) regions of 76 various tumor cell lines were examined to investigate the existence of a specific relationship between a somatic mtDNA sequence and initiation and/or progression of a tumor. Based on molecular cloning-sequencing analysis, a nucleotide sequence in the D-loop region in each cell line was found to be homoplasmic. Several site-specific nucleotide variations were found in stomach and liver tumor cell lines more frequently than those in other tumor cell lines. Subsequently, 20 pairs of noncancerous and cancerous parts from stomach and liver tumor tissues were examined. In the liver tumor tissue, 80% of the noncancerous parts exhibited slightly higher heterogeneity than the corresponding cancerous parts. Several site-specific nucleotide variations found in 76 tumor cell lines were also detected in noncancerous or cancerous parts of stomach and liver tumor tissues. However, it remains unclear why the mtDNA D-loop sequence is homoplasmic in each tumor cell line. The data indicate that mtDNA exhibits heterogeneity even in the noncancerous part and a slight decrease in heterogeneity during tumorigenesis and/or tumor progression. Homoplasmy of the mtDNA population in the tumor cell line would be acquired in the cloning process of establishing a cell line. Site-specific nucleotide substitutions might not be directly involved in the tumorigenesis process.

Interaction of the hepatitis B virus X protein (HBx) with heat shock protein 60 enhances HBx-mediated apoptosis.

Understanding the function of the hepatitis B virus X protein (HBx) is fundamental to elucidating the underlying mechanisms of hepatitis and hepatocarcinogenesis caused by hepatitis B virus (HBV) infection. We identified heat shock protein 60 (Hsp60) as a novel cellular target of HBx by the combination of affinity purification and mass spectrometry. Physical interaction between HBx and Hsp60 was confirmed by standard immunoprecipitation and immunoblot methods. Analysis of HBx deletion constructs showed that amino acids 88-117 of HBx were responsible for the binding to Hsp60. Confocal laser microscopy demonstrated that HBx and Hsp60 colocalized in mitochondria. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) revealed that the introduction of Hsp60 into cells facilitated HBx-induced apoptosis. These findings suggest the importance of the molecular chaperon protein Hsp60 to the function of HBV viral proteins.

PI3K inhibitors changed the p53-induced response of Saos-2 cells from growth arrest to apoptosis.

p53 is activated by stress leading to oncogenic alteration, which induces either cell cycle arrest or apoptosis, although the mechanism involved in this decision has not been fully clarified as yet. This work was undertaken to change the cellular response by inducing apoptosis with PI3K inhibitors to Saos-2 cells that had been growth-arrested in both G1 and G2/M by the wild-type activity of temperature-sensitive (ts) p53. We found that the PI3K/Akt inhibitors LY294002 and wortmannin, but not the MEK inhibitor U0126, were capable of inducing apoptosis in growth-arrested Saos-2 cells, as assessed by an increase in the sub-G1 population, pyknotic nuclei, and DNA ladder formation. We detected the cleavage of caspases 9 and 3, and PARP after LY294002 addition, accompanied by a loss of cytochrome c from the mitochondria, and observed Bax translocation to the mitochondria and down-regulation of phospho-Akt, suggesting that blocking of survival signals triggered the apoptotic signal through the mitochondrial apoptotic pathway. It is thus suggested that the PI3K/Akt pathway played an important role in determining cell fate between growth arrest and apoptosis.

Hepatitis B virus X protein induces cell death by causing loss of mitochondrial membrane potential.

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.

Synergistic activation of the serum response element-dependent pathway by hepatitis B virus x protein and large-isoform hepatitis delta antigen.

Hepatitis delta virus (HDV) is a naturally occurring satellite of hepatitis B virus (HBV). There are few studies of the effects of the combination of HBV and HDV proteins (HDV antigens [HDAgs]) on intracellular signaling pathways. To understand the influence of HBV and HDV coinfection on hepatocytes, we investigated the effect of HBV proteins and HDAgs on the serum response element (SRE)-dependent pathway. Reporter assays revealed that only HBV X protein (HBx), alone or with the large isoform of HDAg (LHDAg), synergistically activated the SRE-dependent pathway. The effect of HBx and LHDAg on Elk1 or serum response factor (SRF) was examined, because both proteins bind to the SRE. HBx activated the transcriptional ability of Elk1, whereas LHDAg activated the transcriptional ability of SRF. Thus, HBx and LHDAg synergistically activated the SRE-dependent pathway. These results may help us understand clinical phenomena in patients coinfected with HBV and HDV.

A new ERK2 binding protein, Naf1, attenuates the EGF/ERK2 nuclear signaling.

Extracellular signal regulated kinase1/2 (ERK1/2), an important factor in signal transduction, controls cell growth, differentiation, and death. To elucidate the details of the mechanism of ERK1/2 signaling in human cells, we isolated Nef-associated factor 1 alpha (Naf1 alpha) by a yeast two-hybrid system, which bound to human ERK2. The binding was confirmed by a pull-down assay in vitro and immunoprecipitation in vivo. Upon EGF treatment, Naf1 alpha was phosphorylated by the EGF/MEK/ERK2 signal transduction pathway. To identify the role of Naf1 alpha in the ERK2 signaling, Naf1 alpha-expressing Saos-2 cells were analyzed for ERK2 nuclear translocation and activation of its downstream target. Overexpression of Naf1 alpha suppressed ERK2 entering into the nucleus and inhibited the ERK2-dependent Elk1-driven luciferase transcription, suggesting Naf1 alpha to be an attenuator of activated ERK2 signaling.