1. Volume 132, Issue 4, Pages 1476-1494 (April 2007)
Aberrant Epigenetic Modifications in Hepatocarcinogenesis Induced by Hepatitis B Virus X Protein 
In Young Park, Bo Hwa Sohn, Eunsil Yu, Dong Jin Suh, Young–Hwa Chung, Je–Ho Lee, Stefan J. Surzycki, Young Ik Lee 
Gastroenterology 
Volume 132, Issue 4, Pages (April 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
HBx modulates the expression of DNMTs. (A) DNMT activity assay of Chang liver (CHL), HepG2, and Huh7 human liver cell lines transiently or stably transfected with pHBx. The transiently transfected DNA quantities were 0.1 μg, 0.5 μg, or 1.0 μg of pHBx DNA per 1 × 106 cells. The observed DNMT activities (mean ± SE) in HBx-expressing cells were normalized to that in mock-transfected CHL cells. *Indicates that the mean values in HBx stably transfected cells are significantly different from those of the mock-transfected controls (P < .05). (B) Expression profiles of DNMTs in pHBx-transfected CHL cells obtained by Northern blot (left) and immunoblot (right) analysis. The transiently transfected DNA quantities were as follows: +, 0.1 μg; ++, 0.5 μg; and +++, 1.0 μg of pHBx DNA per 1 × 106 cells. CHL-X, CHL cells stably transfected with pHBx. (C) Luciferase assay of luciferase reporter constructs driven by DNMT promoters in CHL cells cotransfected with or without pHBx. The ratio of luciferase reporter constructs to pHBx and β-galactosidase was 5:10:1. The observed luciferase activities were normalized to the mean activities of the basal promoters (mean ± SE). *Indicates that the mean values in CHL cells cotransfected with pHBx are significantly different from those of mock-transfected controls (P < .05).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
Chromatin immunoprecipitation (ChIP) of DNMT1 (A), DNMT3A (B), and DNMT3B (C) in CHL cells transfected with pHBx (1 μg per 1 × 106 cells) or with empty vector. The locations of PCR products are indicated as numbers and lines under the simplified genomic structures of the DNMTs. Relative immunoprecipitated band intensities were normalized to the band intensities of PCP products obtained from a known 0.02% input DNA. Data represent the mean ± SE. *Indicates that the mean values in pHBx-transfected cells are significantly different from those of the mock-transfected controls (P < .05). Solid box, exon; arrow, transcription initiation site.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
HBx represses IGFBP-3 expression by de novo DNA methylation. (A) Northern blot and immunoblot analyses of IGFBP-3 expression in pHBx-transfected CHL cells. CHL-X, CHL cells stably expressing HBx; +, 0.1 μg; ++, 0.5 μg; +++, 1.0 μg of pHBx DNA per 1 × 106 cells. (B) IGFBP-3 expression in CHL-X cells after 5-AzaC treatment for 48 hours was determined by quantitative RT-PCR and immunoblot analysis. (C) Luciferase assay of reporter constructs driven by the IGFBP-3 promoter (pBP3-Luc) in CHL, Huh7, and Hep3B cells cotransfected with and without pHBx. In pBP3-Luc/Sss I, the pBP3-Luc DNA was premethylated with Sss I methyltransferase in vitro. The observed activities were normalized to the mean activity of pBP3-Luc (mean ± SE for 4 independent assays). The transfected quantity of pBP3-Luc was 0.25 μg, and those of pHBx were 0.1 μg (+) or 0.5 μg (++) per 1 × 105 cells. (D) Bisulfite sequencing of the endogenous IGFBP-3 promoter region in CHL-X and mock CHL cells from passage 5 (P5). The sequences of the CpG sites between −159 and −112 are shown. (E) Bisulfite sequencing of the endogenous IGFBP-3 promoter region in CHL cells 72 hours after transfection of pHBx. The CpG number was counted from nt −285 to −86. (F) MSP of tumor suppressor genes in CHL cells 72 hours after transient transfection with pHBx.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
DNMT3A1 and DNMT3A2 are responsible for HBx-induced de novo methylation of the IGFBP-3 promoter region. (A) The expressions and catalytic activities of recombinant DNMTs transiently expressed in CHL cells. The anti-DsRed antibody recognizes the recombinant DNMT1, and the anti-HA antibody recognizes recombinant DNMT3A1 and 3A2. The observed DNMT activities were normalized to the mean activity in mock-transfected CHL cells over 4 independent assays (mean ± SE). The transfected quantity of each DNMT expression vector was 1 μg per 1 × 106 cells. (B) Luciferase assay using pBP3-Luc in CHL cells cotransfected with pDNMT1, pDNMT3A1, or pDNMT3A2. The observed activities were normalized to the mean activity of pBP3-Luc over 4 independent assays (mean ± SE). The ratio of luciferase reporter constructs to each expression vector and β-galactosidase was 5:10:1. (C) Bisulfite sequencing of the endogenous IGFBP-3 promoter region in CHL cells 72 hours after transfection with pDNMT3A1 or pDNMT3A2. Open circle, unmethylated; solid circle, methylated. (D) Quantitative RT-PCR analysis of DNMTs in CHL cells transfected with a DNMT3A siRNA mix (left). RT-PCR primers for DNMT3A were designed to detect both DNMT3A1 and DNMT3A2. Quantitative RT-PCR and immunoblot analysis of IGFBP-3 expression of CHL cells transfected with pHBx (1 μg) alone, pHBx (1 μg) nmol/L DNMT3AsiRNA mix, or 100 nmol/L DNMT3A siRNA mix alone (right).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
DNMT3A1, DNMT3A2, and MeCP2 are recruited to the IGFBP-3 promoter region in pHBx-transfected CHL cells. (A) The promoter region of IGFBP-3. The transcription initiation site is denoted as +1. The regions used in the ChIP assay (thin lines) and EMSA (thick line) are underlined. R1 (−281 to −58) and R2 (+45 to +268) encompass the promoter region and exon I, respectively. (B) ChIP assay of the endogenous IGFBP-3 promoter regions (R1 and R2) using an anti-DNMT3A antibody in transiently and stably pHBx-transfected CHL cells (left) or with an anti-HA antibody in pDNMT3A1- or pDNMT3A2-transfected CHL cells (right). Transfected DNA quantities were 0.5 μg (++) pHBx, 1.0 μg (+++) pHBx, 1.0 μg pDNMT3A1, or 1.0 μg pDNMT3A2 per 1 × 106 cells. (C) ChIP analysis in pHBx-transfected CHL cells using antibodies against MeCP2, SP1, and MBD2. (D) EMSA using a labeled 27-bp unmethylated ds-oligonucleotide containing the SP1 binding site (−179 to −153) and the same ds-oligonucleotide in vitro methylated with Sss I methyltransferases. Gel shift assays were performed with nuclear extract (NE) isolated from CHL cells. As a competitor, the nonlabeled ds-oligonucleotide was used. For the determination of specific SP1 binding on the probe, an anti-SP1 antibody was used.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
HBx expression promotes genome-wide hypomethylation. (A) Genomic 5-MeC dot blot analysis using an anti-5-MeC antibody in CHL, Huh7, and HepG2 cells stably transfected with HBx at passage 5 (P5) and passage 30 (P30). Equal quantities of genomic DNA were confirmed by 0.02% methylene blue staining. The dot intensities were normalized to the mean intensity of mock-transfected CHL cells (mean ± SE for 4 independent blots). *Indicates that the mean values in HBx-stable cells at P30 are significantly different from those of the mock-transfected controls (P < .05). (B) Methylation-sensitive Southern blot analysis of a satellite 2 sequence probe (left) in P30 CHL, Huh7, and HepG2 cells stably expressing HBx. A photograph of the gel before membrane transfer is included (right). The increased band intensities in the P30 stable pHBx transfectants are indicated with arrowheads. (C) HBx enhances tumor growth in 10-week-old athymic nude mice injected with ∼1 × 107 CHL cells. The volumes of tumors developed in athymic nude mice were measured every 2 weeks after cell injection. The measured tumor volumes are indicated as mean volumes with standard error bars. (D) Genomic 5-MeC dot blot analysis of tumor tissues derived from the athymic nude mice. The dot intensities were normalized to the mean intensity from the mock-transfected CHL cell-derived tumors, with standard error bars shown for 4 independent blots (mean ± SE).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
Epigenetic abnormalities in HBx-positive HCC patients. (A) Genomic 5-MeC dot blot analysis in 20 tumor and nearby nontumor tissues from HCC patients. HBx expression was examined using RT-PCR and indicated as HBx (+) or (−). The dot intensities were normalized to the mean intensity of normal human liver genomic DNA (mean ± SE). *Indicates that the mean values of HBx-positive tissues are significantly different from HBx-negative tissues (P < .05). N, T, and N.L represent nontumor tissue, tumor tissue, and purchased normal human liver genomic DNA, respectively. (B) MSP analysis of the IGFBP-3 promoter region in tumor and nearby nontumor tissues from HCC patients. U, unmethylated; M, methylated; Not treated, not subjected to bisulfite treatment. (C) RT-PCR analysis of IGFBP-3 in HCC patient tissues. (D) Bisulfite sequencing of the IGFBP-3 promoter region from normal liver, HBx-negative HCC, and HBx-positive HCC tissue genomic DNA. The sequenced region was as described in Figures 3 and 4, −285 to −86 from the transcription initiation site. Open circle, unmethylated; solid circle, methylated CpG.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions