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p300
$40
Journal of Clinical Virology 2006, Vol 36 (suppl 2)
Abstracts, 12th ISHVLD
in people with HCC than those without, and that these mutants are present in nodules of hepatocytes in infected people. These mutants cause stress in the endoplasmic reticulum (ER) and induce the unfolded protein response (UPR) in transfected cells. Many neoplasms, including HCC, show evidence of activation of the UPR, and blocking proteins downstream of the UPR has been shown to block tumor growth in mouse fibrosarcoma models. Therefore, we hypothesized that preS2 mutants are involved in the pathogenesis of HCC. Methods: To determine if preS2 mutants play a causal role in the development of HCC, we generated transgenic mice containing one such mutant genome (Mutant 1). These mice were followed for the development of liver injury and disease. Results: There was no evidence of significant hepatocellular injury in Mutant 1 mice as detected by histological analysis or measurement of serum aspartate aminotransferase levels within the first 14 months of life. However, at 23-25 months of age both homozygous and heterozygous Mutant 1 transgenic mice showed a high incidence of hepatocellular neoplasms, while non-transgenic littermates had only a single small adenoma (Table). Similar to the situation in people, there was a strong male preponderance of hepatocellular neoplasia in Mutant 1 mice. Immunoprecipitation followed by Western blotting revealed similar amount of hepatic X protein as wildtype HBV transgenic mice, ruling out X protein overexpression as the cause of carcinogenesis. The induction of the UPR was confirmed by the splicing of XBP1.
to allow us to introduce HBx into all of cells in a concentrationdependent manner. We observed that HBx and its mutants tested here all enter across cell membrane and overcome the problems generated by DNA transfection experiment such as over-expression and low-efficiency of introduction. In addition, the Big Blue rat cell line system was utilized in combination to reveal the potential mutagenic effect of HBx on the cll gene. HBx increased mutation frequency of the cll gene more than 4-times as compared with that of control in the absence of HBx or that of mutant protein only containing the N-terminus portion (aa 1-67) of HBx. Conclusion: The present study clearly indicates that HBx is able to facilitate DNA mutation via ROS generation in mitochondria. Role of HBx in cell transformation and/or cell death in the cell culture system will be discussed. The major part of this study has been performed at the Cancer Institute, JFCR. **Present address: Material Analysis Division, Japan Patent Office, Chiyoda-ku, Tokyo 1 0 0 4 9 1 5 .
Incidence of hepatocellular neoplasia in mutant 1 mice
Background and Objectives: Hepatitis B virus (HBV) infection is a worldwide health problem and one of the major causes of hepatocellular carcinoma (HCC). Despite epidemiological evidence linking HBV infection and HCC, we poorly understand the mechanisms underlying HBV-associated carcinogenesis. Among HBV proteins, HBx is suspected to contribute to hepatocarcinogenesis. HBx is described as a transcriptional activator that can activate viral promoters as well as a large number of cellular promoters. However, HBx does not directly bind DNA, and rather activates transcription through protein-protein interactions. To better understand the molecular basis for HBx transcriptional activity, we analyzed the effects of HBx on the CREB/basic leucine zipper (bZIP) family of transcription factors, which play pivotal roles in the liver by regulating cellular processes such as gluconeogenesis, lipid metabolism and cell proliferation. Recently, CREB has also been implicated in hepatocarcinogenesis. The aim of our study was to determine whether HBx interacts directly and cooperates with the histone acetyltransferase CBP/p300 and to assess whether this interaction, by upregulating cellular target genes, could be involved in the development of HCC. Methods: We used luciferase reporter assays, in vivo and in vitro binding assays, and chromatin immunoprecipitation (CHIP) assays to investigate HBx activity on CREB-dependent transcription and HBx interaction with CBP/p300. Microarray analysis was used to identify target genes that are regulated by HBx in primary human hepatocytes. Results: We have shown that HBx associates and cooperates with CBP/p300 in CREB-mediated transcriptional activation, and that phosphorylation of CREB is a prerequisite for coactivation of CREB by HBx. Using ChIP assays, we demonstrated that HBx facilitates the formation of CREB/CBP/p300 complexes on target cellular promoter. Finally, a set of candidate target genes of HBx has been identified after transduction of HBx by the lentiviral pTRIP vector into primary human hepatocytes, including genes known to be controlled by CREB, such as PCNA and IL-8. Conclusion: The histone acetyltransferase CBP/p300 plays a crucial role on HBx function in transcriptional activation. To investigate possible mechanisms by which HBx could be involved in hepatocarcinogenesis, we are currently analyzing the interaction of HBx and CBP/p300 on the promoters of HBx target genes using ChIP analysis. Finally, to understand the role of CREB and CBP/p300 in viral replication, we are studying the role of these factors on viral transcription and virion production in HepG2 cells.
Homozygous mutant 1 Heterozygous mutant 1 Non-transgenic littermates
No hepatocellular neoplasia
Hepatocellular adenoma
Hepatocellular carcinoma
7 14 22
0 3 1
6 3 0
Combining adenomas and carcinomas, p < 0.002 using chi-square test for trend.
Conclusion: We have provided the first evidence for HBV directly causing HCC, which appears to result from dysregulated expression of surface proteins from a naturally occurring mutant selected by immune pressure. Our transgenic mice represent a novel model system to understand further the molecular mechanisms of this unusual mode of carcinogenesis, as well cofactors that may contribute to the development of HCC in HBV-infected patients. I-O-_~
Hepatitis B virus X protein causes mutation of cellular gene by ROS generation
K. Koike 1 *, Y. Shirakata 2. 1Institute for Life Sciences, Kitasato
University; 2Department of Gene Research, The Cancer Institute, Tokyo, Japan Background and Objectives: Hepatitis B virus (HBV) chronically infects more than 350 million people worldwide and causes serious liver diseases including cirrhosis and liver cancer, accompanied by hepatocyte death and regeneration. Among the proteins encoded by the HBV genome, the 17-kDa regulatory protein, HBx, has been shown to induce transformation of some rodent cell types and to bring about liver cancer in some transgenic mice. Non-pathogenic HBx transgene increased susceptibility to chemical carcinogenesis. Although HBx itself is unable to bind DNA directly, many reports have shown that HBx activates multiple cellular functions through cis-acting elements such as AP-1, ATF/CREB, NF-kB, etc., proliferative to the cells. HBx also stimulates several signal transduction cascades, including the RAS/MAPK-, NF-kB-, and Src-dependent pathways. On the other hand, we have previously shown using HBx gene transfection that HBx facilitated cell death induction. Methods: (1) Cell culture and cytochemistry, (2) protein transduction, (3) FACS analysis, (4) Western blot analysis, (5) measurement of cellular ATP level, (6) mutation rate analysis using Big Blue rat cell line, (7) DNA sequencing. Results: In addition to the previous data, we revealed that some HBx mutant gene also facilitates cell death induction. The association of HBx with mitochondria causes loss of mitochondrial membrane potential, indicating that HBx has both proliferative and cell death-promoting activities using each different domain structure.To understand the mechanism of HBx-induced cell transformation in more detail, the protein transduction technique has been mobilized
[-O_~
Functional interaction between HBX and the histone acetyltransferase CBP/p300
D. Cougot 1, Y. Wu 1, J. Caramel 1, M. Buendia 1, C. Neuveut 2 *
1Virologie, 20ncogenese et Virologie Mol#culaire, Institut Pasteur, Paris, France
Journal of Clinical Virology 2006, Vol 36 (suppl 2)
Abstracts, 12th ISHVLD
in people with HCC than those without, and that these mutants are present in nodules of hepatocytes in infected people. These mutants cause stress in the endoplasmic reticulum (ER) and induce the unfolded protein response (UPR) in transfected cells. Many neoplasms, including HCC, show evidence of activation of the UPR, and blocking proteins downstream of the UPR has been shown to block tumor growth in mouse fibrosarcoma models. Therefore, we hypothesized that preS2 mutants are involved in the pathogenesis of HCC. Methods: To determine if preS2 mutants play a causal role in the development of HCC, we generated transgenic mice containing one such mutant genome (Mutant 1). These mice were followed for the development of liver injury and disease. Results: There was no evidence of significant hepatocellular injury in Mutant 1 mice as detected by histological analysis or measurement of serum aspartate aminotransferase levels within the first 14 months of life. However, at 23-25 months of age both homozygous and heterozygous Mutant 1 transgenic mice showed a high incidence of hepatocellular neoplasms, while non-transgenic littermates had only a single small adenoma (Table). Similar to the situation in people, there was a strong male preponderance of hepatocellular neoplasia in Mutant 1 mice. Immunoprecipitation followed by Western blotting revealed similar amount of hepatic X protein as wildtype HBV transgenic mice, ruling out X protein overexpression as the cause of carcinogenesis. The induction of the UPR was confirmed by the splicing of XBP1.
to allow us to introduce HBx into all of cells in a concentrationdependent manner. We observed that HBx and its mutants tested here all enter across cell membrane and overcome the problems generated by DNA transfection experiment such as over-expression and low-efficiency of introduction. In addition, the Big Blue rat cell line system was utilized in combination to reveal the potential mutagenic effect of HBx on the cll gene. HBx increased mutation frequency of the cll gene more than 4-times as compared with that of control in the absence of HBx or that of mutant protein only containing the N-terminus portion (aa 1-67) of HBx. Conclusion: The present study clearly indicates that HBx is able to facilitate DNA mutation via ROS generation in mitochondria. Role of HBx in cell transformation and/or cell death in the cell culture system will be discussed. The major part of this study has been performed at the Cancer Institute, JFCR. **Present address: Material Analysis Division, Japan Patent Office, Chiyoda-ku, Tokyo 1 0 0 4 9 1 5 .
Incidence of hepatocellular neoplasia in mutant 1 mice
Background and Objectives: Hepatitis B virus (HBV) infection is a worldwide health problem and one of the major causes of hepatocellular carcinoma (HCC). Despite epidemiological evidence linking HBV infection and HCC, we poorly understand the mechanisms underlying HBV-associated carcinogenesis. Among HBV proteins, HBx is suspected to contribute to hepatocarcinogenesis. HBx is described as a transcriptional activator that can activate viral promoters as well as a large number of cellular promoters. However, HBx does not directly bind DNA, and rather activates transcription through protein-protein interactions. To better understand the molecular basis for HBx transcriptional activity, we analyzed the effects of HBx on the CREB/basic leucine zipper (bZIP) family of transcription factors, which play pivotal roles in the liver by regulating cellular processes such as gluconeogenesis, lipid metabolism and cell proliferation. Recently, CREB has also been implicated in hepatocarcinogenesis. The aim of our study was to determine whether HBx interacts directly and cooperates with the histone acetyltransferase CBP/p300 and to assess whether this interaction, by upregulating cellular target genes, could be involved in the development of HCC. Methods: We used luciferase reporter assays, in vivo and in vitro binding assays, and chromatin immunoprecipitation (CHIP) assays to investigate HBx activity on CREB-dependent transcription and HBx interaction with CBP/p300. Microarray analysis was used to identify target genes that are regulated by HBx in primary human hepatocytes. Results: We have shown that HBx associates and cooperates with CBP/p300 in CREB-mediated transcriptional activation, and that phosphorylation of CREB is a prerequisite for coactivation of CREB by HBx. Using ChIP assays, we demonstrated that HBx facilitates the formation of CREB/CBP/p300 complexes on target cellular promoter. Finally, a set of candidate target genes of HBx has been identified after transduction of HBx by the lentiviral pTRIP vector into primary human hepatocytes, including genes known to be controlled by CREB, such as PCNA and IL-8. Conclusion: The histone acetyltransferase CBP/p300 plays a crucial role on HBx function in transcriptional activation. To investigate possible mechanisms by which HBx could be involved in hepatocarcinogenesis, we are currently analyzing the interaction of HBx and CBP/p300 on the promoters of HBx target genes using ChIP analysis. Finally, to understand the role of CREB and CBP/p300 in viral replication, we are studying the role of these factors on viral transcription and virion production in HepG2 cells.
Homozygous mutant 1 Heterozygous mutant 1 Non-transgenic littermates
No hepatocellular neoplasia
Hepatocellular adenoma
Hepatocellular carcinoma
7 14 22
0 3 1
6 3 0
Combining adenomas and carcinomas, p < 0.002 using chi-square test for trend.
Conclusion: We have provided the first evidence for HBV directly causing HCC, which appears to result from dysregulated expression of surface proteins from a naturally occurring mutant selected by immune pressure. Our transgenic mice represent a novel model system to understand further the molecular mechanisms of this unusual mode of carcinogenesis, as well cofactors that may contribute to the development of HCC in HBV-infected patients. I-O-_~
Hepatitis B virus X protein causes mutation of cellular gene by ROS generation
K. Koike 1 *, Y. Shirakata 2. 1Institute for Life Sciences, Kitasato
University; 2Department of Gene Research, The Cancer Institute, Tokyo, Japan Background and Objectives: Hepatitis B virus (HBV) chronically infects more than 350 million people worldwide and causes serious liver diseases including cirrhosis and liver cancer, accompanied by hepatocyte death and regeneration. Among the proteins encoded by the HBV genome, the 17-kDa regulatory protein, HBx, has been shown to induce transformation of some rodent cell types and to bring about liver cancer in some transgenic mice. Non-pathogenic HBx transgene increased susceptibility to chemical carcinogenesis. Although HBx itself is unable to bind DNA directly, many reports have shown that HBx activates multiple cellular functions through cis-acting elements such as AP-1, ATF/CREB, NF-kB, etc., proliferative to the cells. HBx also stimulates several signal transduction cascades, including the RAS/MAPK-, NF-kB-, and Src-dependent pathways. On the other hand, we have previously shown using HBx gene transfection that HBx facilitated cell death induction. Methods: (1) Cell culture and cytochemistry, (2) protein transduction, (3) FACS analysis, (4) Western blot analysis, (5) measurement of cellular ATP level, (6) mutation rate analysis using Big Blue rat cell line, (7) DNA sequencing. Results: In addition to the previous data, we revealed that some HBx mutant gene also facilitates cell death induction. The association of HBx with mitochondria causes loss of mitochondrial membrane potential, indicating that HBx has both proliferative and cell death-promoting activities using each different domain structure.To understand the mechanism of HBx-induced cell transformation in more detail, the protein transduction technique has been mobilized
[-O_~
Functional interaction between HBX and the histone acetyltransferase CBP/p300
D. Cougot 1, Y. Wu 1, J. Caramel 1, M. Buendia 1, C. Neuveut 2 *
1Virologie, 20ncogenese et Virologie Mol#culaire, Institut Pasteur, Paris, France