Ground glass hepatocytes (GGHs) are the historic hallmarks for the hepatocytes in the late and non-replicative stages of hepatitis B virus (HBV) infection. We have identified type I and type II GGHs that contain two mutant types of large HBV surface antigens (HBsAg) with deletions over the pre-S1 and pre-S2 regions, respectively. These pre-S mutant HBVsAg accumulate in endoplasmic reticulum (ER), resulting in strong ER stress. Type II GGHs often appear in hepatic nodules in the late phases of HBV infection and proliferate in clusters, suggesting that these mutant pre-S1/S2 HBsAg may be involved in HBV-related hepatocarcinogenesis, associated with ER stress. In this study, we investigated the potential genomic instability imposed by pre-S mutant HBsAg. Based on the analysis of comet assays, we found that the pre-S1 and pre-S2 mutant HBsAg caused oxidative stress and DNA damage. The DNA repair gene ogg1 was greatly induced by over-expression of pre-S mutant HBsAg. Induction of the DNA repair gene ogg1 was also detected in the pre-S2 HBsAg transgenic mice, as well as the type II GGHs from patients with hepatocellular carcinoma, strongly suggesting that the pre-S mutant HBsAg contributes to the oxidative DNA damage to hepatocytes. In addition, the mutation rates in the X-linked hprt gene were enhanced in mouse hepatoma ML1-4a cells, which constitutively expressed the pre-S1/S2 HBsAg. These results indicate that pre-S1/S2 mutant HBsAg, which make up GGHs, induce oxidative DNA damage and mutations in hepatocytes in the late stages of HBV infection.

Although hepatitis B virus (HBV) has been documented to cause hepatocellular carcinoma (HCC), the exact role of HBV in the development of HCC remains enigmatic. Several hypotheses have been proposed to explain the potential mechanism, including insertional mutagenesis of HBV genomes and transcriptional activators of HBV gene products such as hepatitis B x protein (HBx) and truncated middle S mutants. In the past few years, we have identified two types of large HBV surface antigens (LHBs) with deletions at the pre-S1 (∆ ∆ ∆ ∆S1-LHBs) and pre-S2 (∆ ∆ ∆ ∆S2-LHBs) regions in ground glass hepatocytes. The pre-S mutant LHBs are retained in the endoplasmic reticulum (ER) and escape from immune attack. The pre-S mutants, particularly ∆ ∆ ∆ ∆S2-LHBs, are increasingly prevalent in patients with hepatitis B e antigen (HBeAg)-positive chronic HBV infection, ranging from 6% before the 3rd decade to 35% in the 6th decade. In HCC patients, the two pre-S mutants were detected in 60% of HCC patients, in the serum and in HCC tissues. Pre-S mutant LHBs can initiate ER stress to induce oxidative DNA damage and genomic instability. Furthermore, pre-S mutant LHBs can upregulate cyclooxygenase-2 and cyclin A to induce cell cycle progression and proliferation of hepatocytes. In transgenic mice, the pre-S mutants can induce dysplasia of hepatocytes and development of HCC. In a nested control study, the presence of pre-S mutants carried a high risk of developing HCC in HBV carriers. In summary, the findings we describe in this review suggest a potential role for HBV pre-S mutants in HBV-related hepatocarcinogenesis, providing a model of viral carcinogenesis associated with ER stress. (Cancer Sci 2006; 97: 683-688) H epatitis B virus is recognized as a major etiological factor in the development of HCC.(1,2) Epidemiological studies have demonstrated an approximately 100-fold increase in the relative risk of HCC among HBV carriers compared to non-carriers.(3) Although the relationship between chronic HBV infection and HCC has been well established, the exact role of HBV in the pathogenesis of HBV-related hepatocarcinogenesis remains to be elucidated.Hepatitis B virus is a partially double-stranded DNA virus containing a genome of 3.2 kb in size, which contains four open reading frames encoding viral polymerase, the core and e antigen, the HBx protein and the pre-S/S gene encoding the three surface antigens (i.e. the large [pre-S1 + pre-S2 + S], middle [pre-S2 + S] and small [S only] surface proteins). In chronic HBV infection, HBV DNA can be integrated into the host genome. Almost all HCC harbor single or multiple copies of integrated HBV DNA.(4,5) The integrated HBV DNA in tumors is usually rearranged and partially deleted. HBV DNA integration has been shown to be a random event and no specific cis-effect has been observed on flanking cellular genes.(6,7) Therefore, HBV DNA integration per se is not considered to be a general mechanism of HBV-related hepatocarcinogenesis. Based on the observations obtained from several isolates of human H...

Expression of mutant proteins or viral infection mayinterfere with proper protein folding activity in the endoplasmic reticulum (ER). Several pathways that maintain cellular homeostasis were activated in response to these ER disturbances. Here we investigated which of these ER stress-activated pathways induce COX-2 and potentially oncogenesis. Tunicamycin and brefeldin A, two ER stress inducers, increased the expression of COX-2 in ML-1 or MCF-7 cells. Nuclear translocation of NF-B and activation of pp38 MAPK were observed during ER stress. I B␣ kinase inhibitor Bay 11-7082 or I B␣ kinase dominant negative mutant significantly inhibited the induction of COX-2. pp38 MAPK inhibitor SB203580 or eIF2␣ phosphorylation inhibitor 2-aminopurine attenuated the nuclear NF-B DNA binding activity and COX-2 induction. Expression of mutant hepatitis B virus (HBV) large surface proteins, inducers of ER stress, enhanced the expression of COX-2 in ML-1 and HuH-7 cells. Transgenic mice showed higher expression of COX-2 protein in liver and kidney tissue expressing mutant HBV large surface protein in vivo. Similarly, increased expression of COX-2 mRNA was observed in human hepatocellular carcinoma tissue expressing mutant HBV large surface proteins. In ML-1 cells expressing mutant HBV large surface protein, anchorage-independent growth was enhanced, and the enhancement was abolished by the addition of specific COX-2 inhibitors. Thus, ER stress due either to expression of viral surface proteins or drugs can stimulate the expression of COX-2 through the NF-B and pp38 kinase pathways. Our results provide important insights into cellular carcinogenesis associated with latent endoplasmic reticulum stress.

The 26S proteasome degrades proteins targeted by the ubiquitin pathway, a function thought to explain its role in cellular processes. The proteasome interacts with the ubiquitin-like N terminus of Rad23, a nucleotide excision repair (NER) protein, in Saccharomyces cerevisiae. Deletion of the ubiquitin-like domain causes UV radiation sensitivity. Here, we show that the ubiquitin-like domain of Rad23 is required for optimal activity of an in vitro NER system. Inhibition of proteasomal ATPases diminishes NER activity in vitro and increases UV sensitivity in vivo. Surprisingly, blockage of protein degradation by the proteasome has no effect on the efficiency of NER. This establishes that the regulatory complex of the proteasome has a function independent of protein degradation.

Mounting evidence supports the involvement of HBV and its gene products in the multistep progression of liver tumorigenesis. 2 A protein-designated HBx has been extensively studied; the data reveal a role of the protein as a transactivator involved in cell growth, apoptosis, DNA damage signals, mitogen-activated protein kinase, and JAK/STAT signaling pathways. 3 Recently, the large surface protein (LHBs) and a C-terminally truncated middle surface protein (MHBs t ) have likewise been recognized as transactivators that share the same mechanism for transcriptional activation. 4,5 This group of activators may trigger a protein kinase C-dependent activation of the c-Raf-1/mitogen-activated protein kinase 2 signal transduction cascade, resulting in the activation of transcription factors such as activator protein 1 and nuclear factor B. The functional activity of these activators is dependent on the cytoplasmic orientation of the pre-S2 region of MHBs t and LHBs that is also related to their intracellular retention. 6,7 Besides the MHBs t , we have previously identified a mutant Abbreviations: ⌬S2-LHBs, mutant with a deletion in the pre-S2 region of the large surface protein; HBV, hepatitis B virus; ER, endoplasmic reticulum; HCC, hepatocellular carcinoma; GGH, ground glass hepatocyte; cDNA, complementary DNA; HH4, nontransformed human hepatocyte cell line; BFA, brefeldin A; VT, vomitoxin; PCNA, proliferating cell nuclear antigen; CDK, From the