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Hypermethylation of Growth Arrest DNA Damage-Inducible Gene 45 β Promoter in Human Hepatocellular Carcinoma

2004; Elsevier BV; Volume: 165; Issue: 5 Linguagem: Inglês

10.1016/s0002-9440(10)63425-6

1525-2191

Weihua Qiu, Bingsen Zhou, Hongzhi Zou, Xiyong Liu, Peiguo Chu, Richard R. Lopez, Jennifer Shih, Christopher Chung, Yun Yen,

Ubiquitin and proteasome pathways

Growth arrest DNA damage-inducible gene 45 β (GADD45β) has been known to regulate cell growth, apoptotic cell death, and cellular response to DNA damage. Down-regulation of GADD45β has been verified to be specific in hepatocellular cancer (HCC) and consistent with the p53 mutant, and degree of malignancy of HCC. This observation was further confirmed by eight HCC cell lines and paired human normal and HCC tumor tissues by Northern blot and immunohistochemistry. To better understand the transcription regulation, we cloned and characterized the active promoter region of GADD45β in luciferase-expressing vector. Using the luciferase assay, three nuclear factor-κB binding sites, one E2F-1 binding site, and one putative inhibition region were identified in the proximal promoter of GADD45β from −865/+6. Of interest, no marked putative binding sites could be identified in the inhibition region between −520/−470, which corresponds to CpG-rich region. The demethylating agent 5-Aza-dC was used and demonstrated restoration of the GADD45β expression in HepG2 in a dose-dependent manner. The methylation status in the promoter was further examined in one normal liver cell, eight HCC cell lines, eight HCC tissues, and five corresponding nonneoplastic liver tissues. Methylation-specific polymerase chain reaction and sequencing of the sodium bisulfite-treated DNA from HCC cell lines and HCC samples revealed a high percentage of hypermethylation of the CpG islands. Comparatively, the five nonneoplastic correspondent liver tissues demonstrated very low levels of methylation. To further understand the functional role of GADD45β under-expression in HCC the GADD45β cDNA constructed plasmid was transfected into HepG2 (p53 WT) and Hep3B (p53 null) cells. The transforming growth factor-β was assayed by enzyme-linked immunosorbent assay, which revealed a decrease to 40% in transfectant of HepG2, but no significant change in Hep3B transfectant. Whereas, Hep3B co-transfected with p53 and GADD45β demonstrated significantly reduced transforming growth factor-β. The colony formation was further examined and revealed a decrease in HepG2-GADD45β transfectant and Hep3B-p53/GADD45β co-transfectant. These findings suggested that methylation might play a crucial role in the epigenetic regulation of GADD45β in hepatocyte transformation that may be directed by p53 status. Thus, our results provided a deeper understanding of the molecular mechanism of GADD45β down-regulation in HCC. Growth arrest DNA damage-inducible gene 45 β (GADD45β) has been known to regulate cell growth, apoptotic cell death, and cellular response to DNA damage. Down-regulation of GADD45β has been verified to be specific in hepatocellular cancer (HCC) and consistent with the p53 mutant, and degree of malignancy of HCC. This observation was further confirmed by eight HCC cell lines and paired human normal and HCC tumor tissues by Northern blot and immunohistochemistry. To better understand the transcription regulation, we cloned and characterized the active promoter region of GADD45β in luciferase-expressing vector. Using the luciferase assay, three nuclear factor-κB binding sites, one E2F-1 binding site, and one putative inhibition region were identified in the proximal promoter of GADD45β from −865/+6. Of interest, no marked putative binding sites could be identified in the inhibition region between −520/−470, which corresponds to CpG-rich region. The demethylating agent 5-Aza-dC was used and demonstrated restoration of the GADD45β expression in HepG2 in a dose-dependent manner. The methylation status in the promoter was further examined in one normal liver cell, eight HCC cell lines, eight HCC tissues, and five corresponding nonneoplastic liver tissues. Methylation-specific polymerase chain reaction and sequencing of the sodium bisulfite-treated DNA from HCC cell lines and HCC samples revealed a high percentage of hypermethylation of the CpG islands. Comparatively, the five nonneoplastic correspondent liver tissues demonstrated very low levels of methylation. To further understand the functional role of GADD45β under-expression in HCC the GADD45β cDNA constructed plasmid was transfected into HepG2 (p53 WT) and Hep3B (p53 null) cells. The transforming growth factor-β was assayed by enzyme-linked immunosorbent assay, which revealed a decrease to 40% in transfectant of HepG2, but no significant change in Hep3B transfectant. Whereas, Hep3B co-transfected with p53 and GADD45β demonstrated significantly reduced transforming growth factor-β. The colony formation was further examined and revealed a decrease in HepG2-GADD45β transfectant and Hep3B-p53/GADD45β co-transfectant. These findings suggested that methylation might play a crucial role in the epigenetic regulation of GADD45β in hepatocyte transformation that may be directed by p53 status. Thus, our results provided a deeper understanding of the molecular mechanism of GADD45β down-regulation in HCC. Hepatocellular carcinoma (HCC) is one of the most common cancers in many parts of the world including the Far East, the southern Sahara, and southern Europe. It accounts for nearly half a million deaths worldwide.1Parkin DM Pisani P Ferlay J Estimates of the worldwide incidence of 25 major cancers in 1990.Int J Cancer. 1999; 80: 827-841Crossref PubMed Scopus (1817) Google Scholar Despite recent advances in diagnostic and therapeutic management, prognosis of HCC remains poor. Recent epidemiological data suggests that the incidence of HCC is increasing in the United States, with the rate of new cases at 17,300 per year.2Jemal A Murray T Samuels A Ghafoor A Ward E Thun MJ Cancer statistics, 2003.Ca Cancer J Clin. 2003; 53: 5-26Crossref PubMed Scopus (3363) Google Scholar Three members of the GADD45 gene family, GADD45α, GADD45β, and GADD45γ, have been identified based on the extensive region of conserved sequence. All of them can be induced by DNA damage and/or other environmental stresses.3Takekawa M Saito H A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK.Cell. 1998; 95: 521-530Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 4Nakayama K Hara T Hibi M Hirano T Miyajima A A novel oncostatin M-inducible gene OIG37 forms a gene family with MyD118 and GADD45 and negatively regulates cell growth.J Biol Chem. 1999; 274: 24766-24772Crossref PubMed Scopus (38) Google Scholar GADD45β was first identified as a myeloid differentiation primary response gene activated in murine myeloid leukemia cells (M1) by interleukin-6 after induction of terminal differentiation. GADD45β has been implicated in regulating cell growth, apoptotic cell death, and cellular responses to DNA damage. As a positive apoptosis modulator, activation of GADD45β prevents the propagation of damaged cells, causing cell growth arrest and subsequent apoptosis after exposure to genotoxins.5Zhang W Bae I Krishnaraju K Azam N Fan W Smith K Hoffman B Leibermann DA CR6: a third member in the MyD118 and GADD45 gene family which functions in negative growth control.Oncogene. 1999; 18: 4899-4907Crossref PubMed Scopus (130) Google Scholar From our previous study, we used the DNA microarray to compare the gene expression profile of HCC tissues with that of matched nonneoplastic tissues. We identified that the expression of GADD45β was significantly down-regulated in HCC. The quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) analyses of 85 HCC cases confirmed the significant and specific GADD45β decrease in HCC. Moreover, down-regulation of GADD45β was strongly correlated with HCC differentiation and advanced nuclear grade.6Qiu W David D Zhou B Chu PG Zhang B Wu M Xiao J Han T Zhu Z Wang T Liu X Lopez R Frankel P Jong A Yen Y Down-regulation of growth arrest DNA damage-inducible gene 45 β expression is associated with human hepatocellular carcinoma.Am J Pathol. 2003; 6: 1961-1974Abstract Full Text Full Text PDF Scopus (67) Google Scholar Our results suggested that the lack of GADD45β expression in HCC might lead to the failure of inhibition of atypical cell growth or apoptosis. Understanding the regulation of GADD45β may reveal the relationship between the process of DNA damage repair and hepatocarcinogenesis. It has been reported that GADD45β can be induced by p53, or growth inhibitory cytokines such as transforming growth factor (TGF)-β.7Yoo J Ghiassi M Jirmanova L Balliet AG Hoffman B Fornace Jr, AJ Liebermann DA Böttinger EP Roberts AB Transforming growth factor-β-induced apoptosis is mediated by Smad-dependent expression GADD45β through p53 activation.J Biol Chem. 2003; 278: 43001-43007Crossref PubMed Scopus (209) Google Scholar, 8Takekawa M Tatebayashi K Itoh F Adachi M Imai K Saito H Smad-dependent GADD45β expression mediates delayed activation of p53 MAP kinase by TGF-β.EMBO J. 2002; 21: 6473-6482Crossref PubMed Scopus (148) Google Scholar The TGF-β-dependent apoptotic pathway is important in the elimination of damaged cells. The proximal promoter of GADD45β is activated by TGF-β through the action of Smad 2, Smad 3, and Smad 4. Expression of GADD45β in AML 12 murine hepatocytes has been shown to induce p38 and trigger apoptosis. Whereas, inhibition of GADD45β expression by anti-sense blocks TGF-β-dependent p53 activation and apoptosis. However, it was known that the hepatoma cell line possesses complex regulation mechanisms such as p53 mutation-dependent or -independent pathway, MAPK or JNK/ERK involve TGF-β-mediated apoptosis, and so forth.7Yoo J Ghiassi M Jirmanova L Balliet AG Hoffman B Fornace Jr, AJ Liebermann DA Böttinger EP Roberts AB Transforming growth factor-β-induced apoptosis is mediated by Smad-dependent expression GADD45β through p53 activation.J Biol Chem. 2003; 278: 43001-43007Crossref PubMed Scopus (209) Google Scholar, 8Takekawa M Tatebayashi K Itoh F Adachi M Imai K Saito H Smad-dependent GADD45β expression mediates delayed activation of p53 MAP kinase by TGF-β.EMBO J. 2002; 21: 6473-6482Crossref PubMed Scopus (148) Google Scholar In this study, we focus on understanding the mechanism of down-regulation of GADD45β. Our result suggests p53-directed methylation of GADD45β may play a role in HCC cell lines and human tissues. The human HCC cell lines HepG2, Hep3B, and normal embryonic liver cell CL-48 were purchased from American Type Culture Collection, Rockville, MD. Other human HCC cell lines SMMC-7721, BEL-7402, BEL-7404, EBL-7405, QGY-7701, and QGY-7703 were purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences as described previously.9Geng CX Zeng ZC Wang JY Docetaxel inhibits SMMC-7721 human hepatocellular carcinoma cells growth and induces apoptosis.World J Gastroenterol. 2003; 9: 696-700PubMed Google Scholar, 10Xu HY Yang YL Gao YY Wu QL Gao GQ Effect of arsenic trioxide on human hepatoma cell line BEL-7402 cultured in vitro.World J Gastroenterol. 2000; 6: 681-687PubMed Google Scholar, 11Wang XW Xie H Presence of Fas and Bcl-2 proteins in BEL-7404 human hepatoma cells.World J Gastroenterol. 1998; 4: 540-543PubMed Google Scholar All tumor cell lines were cultured in high glucose Dulbecco's modified Eagle's medium or RPMI 1640 supplemented with 10% fetal bovine serum and 1% P/S (100 U/ml penicillin and 100 μg/ml streptomycin) at 37°C and 5% CO2. Five pairs of fresh microdissected HCCs and corresponding nonneoplastic liver tissues were retrieved from the surgical pathology files at City of Hope National Medical Center and St. Vincent Hospital. Two fresh HCC tissues with nuclear grade 4 and one fresh HCC tissue with nuclear grade 1 were also included in this profile as the differentiation control. The tissues were examined to confirm the diagnosis and tumor type. Total RNA and genomic DNA were isolated according to the following methods. Procurement of samples was done under institutional human subjects' guidelines and the applicable laws to protect the privacy of patients. Northern blot and IHC study were used to examine the expression of GADD45β from the HCC cell lines and tissues. Total RNA and genomic DNA were isolated using the Rneasy mini kit and the QIAamp DNA mini kit (Qiagen, Valencia, CA). RNA quality was tested by running a 1.2% diethyl pyrocarbonate (DEPC)/MOPS agarose gel and the concentration was measured by UV spectroscopy. RNA was stored in DEPC water with 10 mmol/L dithiothreitol and Rnasin (1 U/ml) at −70°C. The 32P-GADD45β probe and blot conditions were the same as previously described.6Qiu W David D Zhou B Chu PG Zhang B Wu M Xiao J Han T Zhu Z Wang T Liu X Lopez R Frankel P Jong A Yen Y Down-regulation of growth arrest DNA damage-inducible gene 45 β expression is associated with human hepatocellular carcinoma.Am J Pathol. 2003; 6: 1961-1974Abstract Full Text Full Text PDF Scopus (67) Google Scholar The 222-bp probe, including exon 3 of GADD45β, was generated by reverse transcriptase (RT-PCR) from HepG2 total RNA with the following primers: 5′-GGACCCAGACAGCGTGGTCCTCTG-3′ (sense primer, GADD45β +247) and 5′-GTGACCAGGAGACAATGCAGGTCT-3′ (anti-sense primer, GADD45β +445). The SuperScript one-step RT-PCR kit (Invitrogen, Carlsbad, CA) was used for PCR with the following conditions: 1 cycle at 50°C for 30 minutes; 1 cycle at 94°C for 2 minutes; 35 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute; and 1 cycle at 72°C for 10 minutes. The probe was then purified and cleaned using the Gel Extract and Purification kit (Qiagen) and labeled with 32P using the random priming probe kit from Roche (Indianapolis, IN). RNA was electrophoresed in a 1.2% formaldehyde-agarose gel, blotted to a Hybond-N membrane (Amersham, Arlington, IL), and UV-cross-linked. The blots were hybridized for 1 hour at 68°C, washed twice with 2× standard saline citrate/0.1% sodium dodecyl sulfate at room temperature and twice with 0.1× standard saline citrate/0.1% sodium dodecyl sulfate at 60°C. After hybridization, membranes were exposed for 18 hours and then scanned by a phosphorimager. Quantitative analysis was performed using ImageQuant (Molecular Dynamics, Sunnyvale, CA) version 5.0 with GAPDH as a loading control. All experiments were performed in triplicates. In the IHC study, paraffin sections were deparaffinized and blocked in 1:20 normal horse serum. The primary goat anti-human polyclonal GADD45β antibody (200 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) and the biotinylated anti-goat IgG antibody (Vector Laboratories, Burlingame CA) were used as the first and the second antibody. Then, after 45 minutes of incubation with AB Complex (Vector Elite kit, 1:200 dilution; Vector Laboratories), diaminobenzidine [0.05 g diaminobenzidine and 100 μl of 30% H2O2 in 100 ml of phosphate-buffered saline (PBS)], and 1% copper sulfate were applied for 5 and 10 minutes, respectively. Each slide was counterstained with Mayer's hematoxylin. For all IHC studies, PBS was used as a negative control. Granular cytoplasmic stain was accessed as positive. Proximal promoter fragments of GADD45β, spanning −865 to + 6, were cloned upstream of the luciferase gene in the pGL3 basic luciferase expression plasmid (Promega, Madison WI). Six different GADD45β promoter deletion fragments were generated by PCR with GADD45β sense primers: 5′-GGTGAAGCTTGATGTGTATTGGGCTCTTA-3′ (starting at −865), 5′-GAAAGGTACCAGGGGCTGGGGTCGT-3′ (−744), 5′-GGGAAAGCTTCGGTCCGGGACT-3′ (−618), 5′-TTTTAAGCTTTTCTGGCATTCGC-3′ (−470), 5′-GTTCAAGCTTATAAAAGTCGGT-3′ (−273), 5′-CCGAAAGCTTTGGACGAGCGCTCTA-3′ (−123), and an anti-sense primer to + 6 (5′-TATCCTCGCCAAGGACTTTGC-3′). PCR conditions were as follows: 1 cycle at 95°C for 15 minutes; 35 cycles at 94°C for 40 seconds, 60°C for 40 seconds, 72°C for 2 minutes; and 1 cycle at 72°C for 10 minutes. HepG2 genomic DNA was used as the PCR template as previously described.6Qiu W David D Zhou B Chu PG Zhang B Wu M Xiao J Han T Zhu Z Wang T Liu X Lopez R Frankel P Jong A Yen Y Down-regulation of growth arrest DNA damage-inducible gene 45 β expression is associated with human hepatocellular carcinoma.Am J Pathol. 2003; 6: 1961-1974Abstract Full Text Full Text PDF Scopus (67) Google Scholar PCR products were purified and cleaned using Gel Extract and Purification kit (Qiagen) and then inserted into the pDrive cloning vector (Qiagen) according to the manufacturer's protocol. Fragments were then digested from the pDrive using HindIII and inserted into the pGL3 basic vector. Another set of primers were used for the PCR to generate detailed proximal promoter fragments of GADD45β from −618 to −273: the sense primers were 5′-CGGAGGTACCGGGGATTCCAGGCCCCCCCGA-3′ (−591), 5′-CTCGGGTACCGGAAATCCCGCGCGCGCCCGA-3′ (−547), 5′-CCCCGGTACCGCGGCTCGGCTGCCGGGAA-3′ (−520), 5′-CGGCGGTACCGCGCCCTCCTCCCGGTT-3′ (−436), 5′-GCCCGGTACCGCCGCTCCTCCCCCTCCCCTCCG-3′ (−391), 5′-CGCAGGTACCGCTGCACTCGCCCTT-3′ (−348), 5′-CAATGGTACCGGCGAATGACTCCA-3′ (−314), and anti-sense primer to + 6 (5′-CTTCCTCGAGCATGTTGCAATTATAATCCAC-3′). A KpnI site was incorporated into the sense primers and a XhoI site into the anti-sense primer. PCR reactions were performed as above. PCR products were digested by KpnI and XhoI and cleaned by phenol-chloroform extraction and ethanol precipitation. Fragments were then cloned into the corresponding sites of the pGL3 basic plasmid and sequences were confirmed by DNA sequencing. Logarithmically growing HepG2 and CL-48 cells (1 × 106) were transfected with 15 μg of pGL3 promoter luciferase reporter plasmid and 7.5 μg of pSV-β-galactosidase control vector (Promega), which served as an internal control for transfection efficiency. pGL3 basic plasmids, pGL3 enhancer plasmids, and pGL3 promoter plasmids were used for negative and positive controls. Cells were transfected by electroporation in a 4-mm gap cuvette (Eppendorf, Hamburg, Germany) for 80 μs at a voltage of 600 V. Forty-eight hours after electroporation, cells were washed with PBS and harvested by scraping directly into 0.9 ml of reporter lysis buffer (Promega). Protein concentration was measured using the Bio-Rad Bradford assay (Bio-Rad Laboratories, Hercules, CA). The luciferase activity in 20-μl aliquots of cell lysates was measured by luminometry using luciferase reagent (Promega) and β-galactosidase activity was determined using a β-galactosidase assay system (Promega). Promoter activation was determined as the luciferase activity relative to the control after normalizing to β-galactosidase activity. The day before 5-Aza-dC (Sigma Chemical Co., St. Louis, MO) treatment, logarithmically growing cells were seeded at a density of 1 × 105 cells per 10-cm cell culture dish and incubated at 37°C and 5% CO2. On the second day, cells were treated with freshly prepared 5-Aza-dC (10 μmol/L, 50 μmol/L, 100 μmol/L). Forty-eight hours after treatment, the media was removed and cells were washed with PBS. Fresh medium was added, and the cells were incubated for another 48 hours before isolating total cellular RNA and genomic DNA. Total RNA was isolated using the Rneasy mini kit (Qiagen). Running a 1.2% DEPC/MOPS agarose gel tested RNA quality and the concentration was measured by UV spectroscopy. RNA was stored in DEPC water with 10 mmol/L dithiothreitol and Rnasin (1 U/ml) at −70°C. The GADD45β expression was examined by Northern blot as mentioned above. To examine the possibility that lack of GADD45β expression in HCC is associated with hypermethylation of the CpG islands in promoter, MSP was used to locate the hypermethylation area in GADD45β promoter. After the sodium bisulfite treatment, unmethylated cytosine would convert to uracil, but not methylated cytosines.12Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5212) Google Scholar Consequently, uracil would be recognized as thymidine by the Taq polymerase in PCR reaction. Sodium bisulfite-treated DNA (1.0 μg) was used to the MSP amplification using the following primers: the methylation-specific sense primer 5′-TTCGAAAGTTCGGGTCGTTTCGCGC-3′ and anti-sense primer 5′-GGGGACCGAATAAATAACCGCG-3′, which included nucleotides corresponding to potentially methylated Cs; the primers for amplification of unmethylated DNA: sense 5′-AAAGTTTGGGTTGTTTTGTGT-3′ and anti-sense 5′-ACCAAATAAATAACCACA-3′, which included the nucleotides corresponding to C nucleotides changed to T in sense primer and A in anti-sense primer. The genomic DNAs were isolated using QIAamp DNA mini kit (Qiagen) according to the manufacturer's instructions.6Qiu W David D Zhou B Chu PG Zhang B Wu M Xiao J Han T Zhu Z Wang T Liu X Lopez R Frankel P Jong A Yen Y Down-regulation of growth arrest DNA damage-inducible gene 45 β expression is associated with human hepatocellular carcinoma.Am J Pathol. 2003; 6: 1961-1974Abstract Full Text Full Text PDF Scopus (67) Google Scholar For lowering the cross reaction, the annealing temperatures were designed for a 65°C methylation-specific reaction and a 45°C unmethylated reaction. The methylation-specific and unmethylated DNA-specific primers yielded 137-bp and 128-bp PCR products, respectively, both of which covered the third nuclear factor (NF)-κB binding site and the putative inhibition region. The PCR conditions were as follows: 1 cycle at 95°C for 5 minutes; 35 cycles at 94°C for 40 seconds, 60°C (in methylated MSP), or 45°C (in unmethylated MSP) for 40 seconds, 72°C for 2 minutes; and 1 cycle at 72°C for 10 minutes. Genomic DNA (1.0 μg) from the HCC cell lines and tissues was denatured at 42°C for 20 minutes with 2 μl of herring sperm DNA and 5.5 μl of fresh 3 mol/L NaOH for a final volume of 50 μl. Six hundred μl of 4 mol/L bisulfite/10 mmol/L hydroquinone solution (Sigma Chemical Co.) was added to the reaction system. The system was then incubated at 50°C for 16 hours in the dark. The product was cleaned up using a DNA purification kit (Qiagen). Sodium bisulfite-treated DNA was subjected to PCR using the following primers: 5′-TATTTTTAGTAGAATTTGGGAAAGG-3′ (sense primer with start at GADD45β −635) and 5′-CCTCCTATTAATAAAAAAACAAAAAC-3′ (anti-sense primer to GADD45β −330). The conditions for the PCR were as follows: 1 cycle at 95°C for 15 minutes; 35 cycles at 94°C for 40 seconds, 60°C for 40 seconds, 72°C for 2 minutes; and 1 cycle at 72°C for 10 minutes. Then PCR products were cloned into a pDrive vector (Qiagen) via the TA ligation. DNA sequencing with the T7 primer at City of Hope DNA Sequencing Facility confirmed the sequences. Total RNA was isolated from logarithmically growing CL-48 cells with TRIzol reagent (Life Technologies, Rockville, MD). The cDNA was reverse transcribed from total RNA using AMVRT and Oligo(dT)12-18 primer. The DNA fragment of CL-48 was obtained using RT-PCR with the following primers. The forward and reverse oligonucleotide primer: 5′-GGATCCATGACGCTGGAAGAGCTC-3′ (sense primer with start at GADD45β −6) and 5′-GCGGCCGCTCAGCGTTCCTGAAGAGA-3′ (anti-sense primer to GADD45β +490). The 497-bp PCR product included the GADD45β full-length sequence according to access number AF078077 in GenBank. The thermal cycle profile consisted of denaturing at 94°C for 40 seconds, annealing at 55°C for 40 seconds, and extending at 72°C for 2 minutes in 35 cycles. The PCR product was purified and cleaned up in 1.2% agarose gel with ethidium bromide staining using the Gel Extract and Purification kit (Qiagen). After agarose gel purification, the coding sequences for GADD45β was inserted into pDrive cloning vector (Qiagen) via U-A ligation according to protocol. Then, the GADD45β fragment was digested by NotI from pDrive-GADD45β (3′ NotI sticky end was obtained from pDrive vector), and cloned into the fluorescence-expressing vector pIRES2-EGFP (B.D. Clonetech). The correct sequence of pIRES2-EGFP-GADD45β plasmid was confirmed by City of Hope National Medical Center Sequence Lab. Escherichia coli bacteria DH5α was cultured along with the appropriate plasmids in LB media containing 100 μg/ml of ampicillin and shaken overnight at 37°C. Maxiprep was performed using the Qiagen plasmid Mega kit (Qiagen) according to the manufacturer's protocol. HepG2 cells were transfected with pIRES2-EGFP-GADD45β plasmid or pIRES2-EGFP vector by electroporation in a 4-mm gap cuvette (Eppendorf, Hamburg, Germany) using 45 μs/500 V. Hep3B cells were transfected with pIRES2-EGFP vector, pIRES2-EGFP-GADD45β, pp53-EGFP (Clontech, Palo Alto, CA) or pIRES2-EGFP-GADD45β/pp53-EGFP by electroporation using 80 μs/650 V. Forty-eight hours after transfection, cells were washed with PBS and collected for flow cytometry and the supernatant was collected using the TGF-β1 Human, Biotrak ELISA system (Amersham Biosciences Corp., Piscataway, NJ) for TGF-β ELISA assay. Above cells were seeded in six-well plates (1000 cells per well) and incubated for 14 days to allow colonies to develop. The medium was then removed and washed with PBS, then fixed and stained with 50% ethanol and 50% methylene blue for 1 hour. The number of stained colonies was scored and counted with Eagle Eye II (Stratagene, La Jolla, CA). The percentage of colony formation was normalized to colonies formed after transfection with empty pIRES2-EGFP vector. All of the experiments were performed in triplicate. In our previous study, we demonstrated that there is a significant down-regulation of GADD45β in HCC cell lines HepG2 and Hep3B.6Qiu W David D Zhou B Chu PG Zhang B Wu M Xiao J Han T Zhu Z Wang T Liu X Lopez R Frankel P Jong A Yen Y Down-regulation of growth arrest DNA damage-inducible gene 45 β expression is associated with human hepatocellular carcinoma.Am J Pathol. 2003; 6: 1961-1974Abstract Full Text Full Text PDF Scopus (67) Google Scholar To further validate the down-regulation of GADD45β in HCC cell lines, a panel of HCC cell lines including SMMC-7721, BEL-7402, BEL-7404, EBL-7405, QGY-7701, and QGY-7703 were examined. The GADD45β expression in the panel of cell lines was examined by Northern blot. As expected, there was a decrease in the GADD45β RNA observed in a majority of HCC cell lines; whereas, in the HCC cell line QGY-7701 and normal embryonic liver cell line CL-48 expression of GADD45β was easily detected (Figure 1A, top). To assess the transcriptional level of GADD45β in human tissues, eight fresh HCC samples along with five corresponding nonneoplastic liver samples were investigated in this study. Using hematoxylin and eosin staining, the HCC samples were examined. Patients 6, 7, and 8 demonstrated poorly differentiated and well-differentiated tumors, which were used as controls. The samples from patient 1 to 5 were moderately differentiated with nuclear grade 2 to 3. The Northern blot results showed that GADD45β exhibited very strong expression in five non-HCC liver tissues as opposed to matched HCC tissues (Figure 1A, Pt 1 to Pt 5). The HCC samples from patients 6 and 7 showed decrease expression of GADD45β in Northern blot analysis. As the positive control, the tissue from patient 8 showed very high GADD45β expression. In the IHC study, the staining pattern of GADD45β is the diffusive brownish cytoplasmic tint. The GADD45β immunostaining was predominantly localized in the nonneoplastic hepatic cells. The difference in distribution between GADD45β staining in the HCC area and nonneoplastic liver tissue was noticeable (Figure 1B). Together, these cell lines and tissues constituted a panel of GADD45β-expressing and not expressing cells and tissues for additional analyses to define the mechanism of specific loss of GADD45β expression in HCC. The low expression of GADD45β in HCC suggests that HCC cells may lack a proper response to DNA damage, fail of inhibiting atypical cell growth, and trigger apoptosis.6Qiu W David D Zhou B Chu PG Zhang B Wu M Xiao J Han T Zhu Z Wang T Liu X Lopez R Frankel P Jong A Yen Y Down-regulation of growth arrest DNA damage-inducible gene 45 β expression is associated with human hepatocellular carcinoma.Am J Pathol. 2003; 6: 1961-1974Abstract Full Text Full Text PDF Scopus (67) Google Scholar One potential mechanism for the specific down-regulation of GADD45β in HCC could be the inability of HCC cells to respond appropriately because of defects in the normal transcription process or the presence of inhibiting factors. To better understand the possible regulatory mechanisms of GADD45β in HCC, we started with the identification of GADD45β proximal promoter region. GADD45β proximal promoter fragments were cloned into a luciferase reporter system, transfected into HepG2 cells, and assessed for their ability to activate luciferase expression. Because HepG2 is a GADD45β-nonexpressing cell, it is not very suitable for the functional localization of the promoter regions. Therefore, we examined the activity of the promoter luciferase reporter in GADD45β-expressing liver cell CL-48. As shown in Figure 2, activation occurred with the GADD45β promoter fragment spanning −865 to −314 in HepG2 and CL-48. Deletion of this fragment from the 5′-end gradually led to decreased promoter activity. In particular, the removal of 27 bp from the −618 fragment caused a significant decrease in promoter activity. Additional truncations led to a slight but insignificant change in promoter activity from −591 to −520. Interestingly, the promoter activity peak appeared with the deletion until −470, which suggested that there exists a putative inhibition region between −520 and −470. In the next analysis of the region from −470 to −314 was examined. We found that the deletion of as few as 34 bp from the 5′-end of this region led to a 24-fold decrease in promoter activity, whereas further truncation from −436 did not influence promoter activity. Also shown in Figure 2, the promoter activity pattern in CL-48 is almost the same as that in HepG2, with the exception of regions −520/−470, −470/−436 and −348/−314. However, only slightly higher promoter activity could be observed in CL-48 in those areas, which suggests other regulatory mechanisms in addition to transcriptional regulation affecting the differential expression between HCC cell and normal liver cell. A search of the TRANSFAC database identified one putative NF-κB binding site (−602 to −593) and one putative E2F-1 binding site (−452 to −444). Both are located in the most significant promoter activity region, −618/−591 and −470/−436. Two other putative NF-κB binding sites between −591 and −520 (−581 to −572 and −537 to −528) were also found with a relatively low score in the database. The NF-κB binding sites have also been identified in a recent report by another group.13Jin R De Smaele E Zazzeroni F Nguyen DU Papa S Jones J Cox C Gelinas C Franzoso G Regulation of the gadd45beta promoter by NF-kappaB.DNA Cell Biol. 2002; 21: 491-503Crossref PubMed Scopus (67) Google Scholar In the putative inhibitor region −520/−470, no marked putative binding sites could be identified. However, the high percentage of CpG islands in this area can be confirmed, which aroused the hypothesis that methylation could be the reason of specific lack of GADD45β expression in HCC. Altogether, these results suggest that several sites in the proximal promoter could be important in the regulation of GADD45β expression. The putative inhibition region may be related to the CpG islands and methylation. In view of the above promoter analysis, we hypothesized that promoter methylation in putative inhibitor region (−520/−470) may underlie the down-expression of GADD45β in HCC. CpG-rich regions were defined as stretches of DNA with a >50% G+C content, and a >0.6 frequency of observed/expected CpG dinucleotides. Analysis of the GADD45β proximal promoter from −1255 to +6 showed that −1255/1024 and −226/+6 lacked CpG islands. In contrast, the −1025/−225 fragment was a CpG rich region, particularly −625/−425, which had a 65.5% G+C content and a 1.22 frequency of observed/expected CpG. Interestingly, this area covered exactly the putative active promoter region, including three NF-κB consensus binding sites, the E2F-1 consensus binding site and the putative inhibition region. This coincidence further suggested the positive correlation between promoter regulation and hypermethylation. Thus, we focused on the analysis of methylation of this area as a potential mechanism of the down-regulation of GADD45β in HCC. To assess the possible specific role of the hypermethylation of GADD45β promoter's lack of expression in HCC, the demethylation agent 5-Aza-dC14Ferguson AT Evron E Umbricht CB Pandita TK Chan TA Hermeking H Marks JR Lambers AR Futreal PA Stampfer MR Sukumar S High frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silencing in breast cancer.Proc Natl Acad Sci USA. 2000; 97: 6049-6054Crossref PubMed Scopus (420) Google Scholar, 15Widschwendter M Berger J Hermann M Muller HM Amberger A Zeschnigk M Widschwendter A Abendstein B Zeimet AG Daxenbichler G Marth C Methylation and silencing of the retinoic acid receptor-β2 gene in breast cancer.J Natl Cancer Inst. 2000; 92: 826-832Crossref PubMed Scopus (272) Google Scholar, 16Santini V Kantarjian HM Issa JP Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications.Ann Intern Med. 2001; 134: 573-586Crossref PubMed Scopus (367) Google Scholar was used to induce GADD45β expression in HepG2 and CL-48 cells. As shown by Northern blot (Figure 3), expression of GADD45β was low in HepG2 cells and could be significantly induced by 5-Aza-dC treatment in a dose-dependent manner. There was an approximate 2.5-fold increase in GADD45β mRNA with 10 μmol/L 5-Aza-dC, a fourfold increase with 50 μmol/L 5-Aza-dC, and a 4.3-fold increase with 100 μmol/L 5-Aza-dC. Also shown in Figure 3, expression of GADD45β was higher in CL-48, but could not be induced by 5-Aza-dC treatment apparently. These results strongly implicated hypermethylation of the promoter in the specific down-regulation of GADD45β in HCC. To further assess whether the lack of GADD45β expression in HCC may be the result of promoter hypermethylation, we designed the MSP primers to amplify the PCR products by using methylated or unmethylated DNA as templates. The methylated PCR product could be detected in most of HCC cell lines, whereas no PCR product could be detected in unmethylated specific primers system (Figure 4A). In contrast, the normal liver cell CL-48 that has high level of GADD45β expression showed a lack of apparent methylated PCR products. Meanwhile, the PCR products were amplified both by the methylation-specific and unmethylated primers in GADD45β-expressing HCC cell strain QGY-7701. These results indicated the notable hypermethylation status in most of GADD45β-nonexpressing HCC cell lines and partial methylation in QGY-7701, compared with undetectable methylation in CL-48. In eight HCC samples analyzed, seven HCC samples lacked GADD45β expression (patient 1 to patient 7). Among these seven samples, all could amplify the PCR products with methylation-specific primers except patient 6. Meanwhile, weak PCR products were identified in the unmethylated PCR system, which suggested the existence of some unmethylated DNA in HCC tissues. The only samples having high GADD45β expression was patient 8, and the MSP results showed the specific amplification of unmethylated DNA, rather than methylated DNA. As the control, we analyzed the methylation pattern of five nonneoplastic liver tissues, which corresponded with patient 1 to patient 5 HCC tissues. When unmethylated specific primers were used in the MSP system, the strong PCR products were observed in all five samples. In the methylated PCR system, no methylated DNA was detectable in three samples. Very weak methylated products were found in the other two samples (patient 3 and patient 5), which indicated the existence of the partial hypermethylation in nonneoplastic liver tissues. Taken together, hypermethylated status could be identified in a majority of GADD45β-nonexpressing HCC cell lines and six HCC samples with low levels of GADD45β. To further confirm the CpG methylation pattern in GADD45β promoter, we sequenced the sodium bisulfite-treated genomic DNA. The PCR products amplified from sodium bisulfite-treated DNA were cloned into the pDrive plasmid using the TA clone. Positive clones were sequenced to examine the methylated cytosine residues, which would not convert to uracil. The promoter region (−618/−436), containing three NF-κB consensus binding sites, one E2F-1 consensus binding site, and the putative inhibition region, was amplified by PCR using sodium bisulfite genomic DNA as templates. This region spanned 36 CpG dinucleotides sites. Most of the methylation was identified in the 15 CpG dinucleotides located in region −565/−485, which covered the third NF-κB consensus-binding site and the putative inhibition region. The results are summarized in Figure 4, B and C. The sequencing results showed most of the 13 CpG dinucleotides in this area were invariably methylated in GADD45β-nonexpressing HCC cell lines. Partial and very low levels of methylation were also demonstrated in QGY-7701 and CL-48, respectively (Figure 4B). Six HCC samples (patients 1 to 5, and patient 7) with low GADD45β expression showed a high percentage of methylation across this region. However, patient 6 had a low expression of GADD45β and low levels of methylation in HCC tissues, while patient 8 had a high expression of GADD45β with low levels of methylation in HCC tissue. The corresponding nonneoplastic tissues (patients 1 to 5) also showed partial methylation in very low percentages (Figure 4C). No marked methylation was noticed in the first two NF-κB consensus-binding sites and the E2F-1 consensus-binding site. All those results strongly implicated the relationship between hypermethylation in promoter and the low-expression of GADD45β in HCC.