The Induction of Growth Arrest DNA Damage-Inducible Gene 45 β in Human Hepatoma Cell Lines by S-Adenosylmethionine
2007; Elsevier BV; Volume: 171; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.070121
ISSN1525-2191
AutoresWeihua Qiu, Bingsen Zhou, Peiguo Chu, Frank Luh, Yun Yen,
Tópico(s)Epigenetics and DNA Methylation
ResumoDown-regulation of GADD45β, which is known to influence cell growth control, apoptosis, and cellular response to DNA damage, has been verified to be specific in hepatocellular carcinoma and consistent with the degree of malignancy. Here, we identified promoter elements for several transcriptional factors in the proximal promoter of GADD45β using the luciferase assay. As a methyl donor for biological transmethylation reactions, S-adenosylmethionine (SAMe) could restore GADD45β expression in HepG2 in Northern blot analyses and quantitative real-time polymerase chain reaction. Activity and binding capacity of nuclear factor (NF)-κB were confirmed to be specifically induced by SAMe, as evidenced by electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, and a decrease of IκBα in Western blot analyses. The most upstream NF-κB-binding site was crucial for transcriptional activation. In contrast to NF-κB, although there is an E2F-1-binding site adjacent to the NF-κB sites, treatment with SAMe could not induce E2F-1-binding activity. Despite showing a similar GADD45β promoter regulatory pattern as HepG2 (p53 wild type), Hep3B (p53-null) did not exhibit GADD45β induction by SAMe, and the induction could be partially recovered on reconstituting p53 in Hep3B. Thus, our results suggest that GADD45β induction by SAMe via NF-κB may represent a novel mechanism of SAMe-mediated hepatoprotection, with p53 playing an important role. Down-regulation of GADD45β, which is known to influence cell growth control, apoptosis, and cellular response to DNA damage, has been verified to be specific in hepatocellular carcinoma and consistent with the degree of malignancy. Here, we identified promoter elements for several transcriptional factors in the proximal promoter of GADD45β using the luciferase assay. As a methyl donor for biological transmethylation reactions, S-adenosylmethionine (SAMe) could restore GADD45β expression in HepG2 in Northern blot analyses and quantitative real-time polymerase chain reaction. Activity and binding capacity of nuclear factor (NF)-κB were confirmed to be specifically induced by SAMe, as evidenced by electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, and a decrease of IκBα in Western blot analyses. The most upstream NF-κB-binding site was crucial for transcriptional activation. In contrast to NF-κB, although there is an E2F-1-binding site adjacent to the NF-κB sites, treatment with SAMe could not induce E2F-1-binding activity. Despite showing a similar GADD45β promoter regulatory pattern as HepG2 (p53 wild type), Hep3B (p53-null) did not exhibit GADD45β induction by SAMe, and the induction could be partially recovered on reconstituting p53 in Hep3B. Thus, our results suggest that GADD45β induction by SAMe via NF-κB may represent a novel mechanism of SAMe-mediated hepatoprotection, with p53 playing an important role. Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the most common cancer in some geographic areas, particularly in the Far East, South Sahara, and southern Europe. Recent epidemiological data suggest that the incidence of HCC is increasing in Western countries.1Williams R Global challenges in liver disease.Hepatology. 2006; 44: 521-526Crossref PubMed Scopus (661) Google Scholar In many cases, HCC is known to result from environmental exposures such as hepatitis virus, alfatoxin, alcohol, or other in vivo or in vitro genotoxins. Human HCC-prone disorders, such as liver cirrhosis, share a very close relationship to genotoxic DNA damage and mutations of known DNA repair genes.2Nakabeppu Y Sakumi K Sakamoto K Tsuchimoto D Tsuzuki T Nakatsu Y Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids.Biol Chem. 2006; 387: 373-379Crossref PubMed Scopus (218) Google Scholar However, the data to support a role for DNA damage in hepatocarcinogenesis are still quite limited. Growth arrest DNA damage-inducible gene 45 β (GADD45β, also named MyD118) gene was first identified as a myeloid differentiation primary response gene activated by interleukin-6 in the mouse myeloid leukemia cell line M1 on induction of terminal differentiation, which has been implicated in regulating cell growth, apoptotic cell death, and cellular responses to DNA damage.3Abdollahi A Lord KA Hoffman-Liebermann B Liebermann DA Sequence and expression of a cDNA encoding MyD118: a novel myeloid differentiation primary response gene induced by multiple cytokines.Oncogene. 1991; 6: 165-167PubMed Google Scholar In our previous study, we have demonstrated that GADD45β was underexpressed in HCC specifically and significantly. More importantly, we observed that down-regulation of GADD45β was strongly correlated with HCC-poor differentiation and advanced nuclear grade.4Qiu 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 45beta expression is associated with human hepatocellular carcinoma.Am J Pathol. 2003; 162: 1961-1974Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar Our results suggested that the specific lack of GADD45β expression might play an important role in hepatocarcinogenesis. Although hypermethylation in proximal promoter of GADD45β was confirmed in our previous study, the molecular basis of GADD45β down-regulation in HCC was far from clear. Several transcriptional regulatory regions containing nuclear factor (NF)-κB- and E2F-1-binding areas were also identified by means of luciferase assay, but functional evidence and transcriptional regulation mechanism need further elucidation.5Qiu W Zhou B Zou H Liu X Chu PG Lopez R Shih J Chung C Yen Y Hypermethylation of growth arrest DNA damage-inducible gene 45 beta promoter in human hepatocellular carcinoma.Am J Pathol. 2004; 165: 1689-1699Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar S-Adenosylmethionine (SAMe) is an essential compound in cellular transmethylation reactions and serves as a methyl donor in numerous metabolic reactions. SAMe is an important precursor in the synthesis of polyamines and glutathione, the main cellular antioxidants in the liver.6McMillan JM McMillan DC S-Adenosylmethionine but not glutathione protects against galactosamine-induced cytotoxicity in rat hepatocyte cultures.Toxicology. 2006; 222: 175-184Crossref PubMed Scopus (19) Google Scholar In liver injury or chronic liver diseases, the synthesis of SAMe is impaired, which might lead to de-differentiation of hepatocytes with increased regeneration and malignant transformation.7Duong FH Christen V Filipowicz M Heim MH S-Adenosylmethionine and betaine correct hepatitis C virus induced inhibition of interferon signaling in vitro.Hepatology. 2006; 43: 796-806Crossref PubMed Scopus (72) Google Scholar SAMe administration attenuates experimental liver damage, improves survival of patients with alcoholic cirrhosis, and prevents experimental hepatocarcinogenesis.8Bergheim I McClain CJ Arteel GE Treatment of alcoholic liver disease.Dig Dis. 2005; 23: 275-284Crossref PubMed Scopus (57) Google Scholar, 9Wang X Cederbaum AI S-Adenosyl-l-methionine attenuates hepatotoxicity induced by agonistic Jo2 Fas antibody following CYP2E1 induction in mice.J Pharmacol Exp Ther. 2006; 317: 44-52Crossref PubMed Scopus (29) Google Scholar, 10Lu SC Mato JM Role of methionine adenosyltransferase and S-adenosylmethionine in alcohol-associated liver cancer.Alcohol. 2005; 35: 227-234Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar Although there is accumulating evidence on the protective potential of SAMe in the preservation of liver function, the molecular mechanism of SAMe's hepatoprotection is primarily unidentified and needs further exploration. In this study, based on the significant induction of GADD45β by SAMe, binding capacity and activity of transcriptional regulators in proximal promoter were investigated using luciferase assay, electrophoretic mobility shift assay (EMSA), Western blot, and enzyme-linked immunosorbent assay (ELISA). With the help of a special set of research models, HepG2 (p53 wild type) and Hep3B (p53-null), the role of p53 in transcriptional regulation was also analyzed by p53 transfection. The human hepatoma cell lines HepG2 and Hep3B and normal human embryonic liver cell line CL-48 were purchased from American Type Culture Collection (Rockville, MD) and cultured in high-glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% P/S (100 U/ml penicillin and 100 μg/ml streptomycin) at 37°C and 5% CO2. The day before SAMe (Knoll Farmaceutici S.P.A., Milan, Italy) treatment, logarithmically growing cells were seeded at a density of 1 × 106 cells per 25-cm cell culture dish. On the 2nd day, cells were treated with SAMe (0, 0.5, and 1.0 mmol/L). Forty-eight hours after treatment, media were removed, and cells were washed with phosphate-buffered saline (PBS). Total RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, CA). RNA quality was tested by running on a 1.2% diethyl pyrocarbonate/3-(N-morpholino)propanesulfonic acid agarose gel, and the concentration was measured by UV spectroscopy. RNA was stored in diethyl pyrocarbonate water with 10 mmol/L dithiothreitol and RNasin (1 U/ml) at −70°C. We have previously reported that the low-expression of GADD45β was specific to HCC and was consistent with the degree of malignancy of HCC. In this study, Northern blot and quantitative real-time polymerase chain reaction (PCR) were used to examine GADD45β expression change after SAMe treatment. In Northern blot, preparation of 32P-labeled GADD45β probe and blot conditions were the same as previously described.4Qiu 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 45beta expression is associated with human hepatocellular carcinoma.Am J Pathol. 2003; 162: 1961-1974Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar In brief, the 222-bp probe, including exon 3 of GADD45β, was generated by reverse transcriptase-PCR with the following primers: 5′-GGACCCAGACAGCGTGGTCCTCTG-3′ (sense primer, GADD45β +247) and 5′-GTGACCAGGAGACAATGCAGGTCT-3′ (anti-sense primer, GADD45β +445). The probe was purified using a gel extract and purification kit (Qiagen) and labeled with 32P using the random priming probe kit from Roche (Indianapolis, IN). After isolation from HepG2 and Hep3B with or without treatment, total 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 and washed with 2× standard saline citrate/0.1% sodium dodecyl sulfate and 0.1× standard saline citrate/0.1% sodium dodecyl sulfate at different temperature. After hybridization, membranes were exposed to phosphorimager screen for 18 hours and then read by phosphorimager scanner. Quantitative analysis was performed using ImageQuant version 5.0 (Molecular Dynamics, Sunnyvale, CA) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. All experiments were performed in triplicates. In quantitative real-time PCR analyses, total RNA was treated with RNA-free DNase I (Promega, Madison, WI) and reversely transcribed to cDNA using AMV-RT and Oligo(dT)12-18 primer. GADD45β was amplified and detected using the following TaqMan probe and primers: 5′-GGGTGTACGAGTCGGCCAA-3′ (forward), 5′-TGGCCAAGAGGCAGAGGA-3′ (reverse), and 5′-FAM-TTGATGAATGTGGACCCAGACAGCGTG-TAMRA-3′ (probe). The predeveloped TaqMan assay reagents control kit (Perkin-Elmer Applied Biosystems, Foster City, CA) was used to detect GAPDH as an internal control. GADD45β-pEGFP and GAPDH-pT7T3D-PAC plasmids were used as positive controls to generate the standard curve. PCR reaction system was as described previously (Ref. 5Qiu W Zhou B Zou H Liu X Chu PG Lopez R Shih J Chung C Yen Y Hypermethylation of growth arrest DNA damage-inducible gene 45 beta promoter in human hepatocellular carcinoma.Am J Pathol. 2004; 165: 1689-1699Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar): included cDNA, 5 pmol of each primer, 10 pmol of probe, and TaqMan Universal PCR Master Mix. The PCR conditions were as follows: one cycle of 50°C for 2 minutes, 95°C for 10 minutes, and 35 cycles of 95°C for 15 seconds, 60°C for 60 seconds. Each data point was performed in duplicates. In our previous study, we identified the active promoter region of GADD45β and the possible regulation mechanism. Based on marked induction of GADD45β by SAMe, we further examined the change of proximal promoter activity to provide functional evidence for promoter regulation hypotheses. Luciferase reporter GADD45β proximal promoter deletion plasmids were constructed as previously described.5Qiu W Zhou B Zou H Liu X Chu PG Lopez R Shih J Chung C Yen Y Hypermethylation of growth arrest DNA damage-inducible gene 45 beta promoter in human hepatocellular carcinoma.Am J Pathol. 2004; 165: 1689-1699Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar In brief, CL-48 genomic DNA was used as a PCR template. Two GADD45β promoter deletion fragments, spanning −618 to +6, were generated by PCR with sense primers: 5′-GGGAAAGCTTCGGTCCGGGACT-3′ (−618), 5′-TTTTAAGCTTTTCTGGCATTCGC-3′ (−470), and anti-sense primer 5′-TATCCTCGCCAAGGACTTTGC-3′ (+6). PCR products were purified and cleaned up using a gel extract and purification kit (Qiagen) and then cloned into the pDrive cloning vector (Qiagen) via U-A nucleotide matching. After digestion from pDrive plasmids using HindIII, those sequences with another seven fragments were cloned upstream of the luciferase gene in the pGL3 basic luciferase expression plasmid (Promega) via corresponding enzyme digestion sites. Another seven detailed proximal promoter fragments covering active promoter region from −618 to −273 were generated by PCR with the following primers: 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′). Because KpnI site and XhoI enzyme digestion sites were incorporated into sense primers and anti-sense primer, respectively, promoter fragments were obtained by KpnI and XhoI digestion. After cleaning by phenol-chloroform extraction and ethanol precipitation, seven fragments (−591/+6, −547/+6, −520/+6, −436/+6, −391/+6, −348/+6, and −314/+6) were cloned into the corresponding sites of the pGL3 basic plasmid. DNA sequencings were confirmed in the City of Hope National Medical Center Sequence Laboratory. The transfection of reporter plasmids was performed as described before.5Qiu W Zhou B Zou H Liu X Chu PG Lopez R Shih J Chung C Yen Y Hypermethylation of growth arrest DNA damage-inducible gene 45 beta promoter in human hepatocellular carcinoma.Am J Pathol. 2004; 165: 1689-1699Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar In brief, 15 μg of pGL3 promoter luciferase reporter plasmids were transfected into HepG2 and Hep3B with 7.5 μg of pSV-β-galactosidase control vector (Promega). Cells were transfected by electroporation in a 4-mm gap cuvette (Eppendorf, Hamburg, Germany). The electroporation parameter was 50 μs at 600 V for HepG2 and 80 μs at 650 V for Hep3B. SAMe was administrated to cells 48 hours after transfection. After an additional 48 hours of culture, cells were harvested by scraping directly into 0.9 ml of reporter lysis buffer (Promega). With standardized protein concentration, the luciferase activity in 20-μl aliquots of cell lysates was measured then by luminometry using luciferase reagent (Promega). 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. HepG2 and Hep3B without SAMe treatment were included as reaction control. pGL3 basic plasmid, pGL3 enhancer plasmid, and pGL3 promoter plasmids were also included for system controls. HepG2 and Hep3B cells (1× 107) with or without SAMe were washed in ice-cold PBS and suspended in 800 μl of solution A (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L ethylenediamine tetraacetic acid, 0.1 mmol/L ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1 mmol/L dithiothreitol, and 0.5 mmol/L phenylmethyl sulfonyl fluoride) for 15 minutes. Fifty μl of Nonidet P-40 was added, and cell pellets were harvested and vortexed for 10 seconds. After centrifugation at 10,000 rpm for 30 seconds at 4°C, the cell pellets were resuspended in 100 μl of solution B (20 mmol/L HEPES, pH 7.9, 0.4 mol/L NaCl, 1 mmol/L ethylenediamine tetraacetic acid, 1 mmol/L ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1 mmol/L dithiothreitol, and 1 mmol/L phenylmethyl sulfonyl fluoride) and vigorously rocked for 15 minutes at 4°C. Cell lysates were centrifuged (10,000 rpm, 15 minutes, 4°C), and then the supernatant was collected (∼55 μl) and stored at −80°C. Protein concentration was measured using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Human oropharyngeal carcinoma KB cells and human prostate cancer PC3 cells (both were purchased from American Type Culture Collection) were cultured in RPMI 1640 medium with 10% fetal bovine serum and 1% P/S used for non-HCC cell line control. The treatment of SAMe and nuclear protein extraction were performed in the same way as mentioned above. Three NF-κB- and one E2F-1 9-binding sites were found in the GADD45β proximal promoter (positions −602/−593, −581/−572, −537/−528, and −452/−444). Because identification of NF-κB and E2F-1 were based on consensus sequences and high score in TRANSFAC database alignment search, electrophoretic mobility shift assay (EMSA), and super shift assay were used to characterize further the functional evidence for these sites in this study. Five oligonucleotide probes for putative NF-κB-binding sequences and two probes for E2F-1 were designed and synthesized by the City of Hope National Medical Center DNA/RNA/peptide synthesis facility. As shown in Figure 4A, probe 1 contained the first and second NF-κB-binding sites. The first site was mutated in probe 2, whereas the second site was mutated in probe 3. Both sites were mutated in probe 4, and probe 5 included only the second NF-κB site. Sequences of the NF-κB probes were as follows: probe 1: 5′-TCCGGGACTCTCCGCGGATCGGGAGGGGATTCCAGG-3′; probe 2: 5′-TCCTATTCTCTCCGCGGATCGGGAGGGGATTCCAGG-3′; probe 3: 5′-TCCGGGACTCTCCGCGGATCGGGAATCCATTCCAGG-3′; probe 4: 5′-TCCTATTCTCTCCGCGGATCGGGAATCCATTCCAGG-3′; probe 5: 5′-TCGCGCGCTGGAAATCCCGCG-3′. The two probes used for E2F-1 (wild type and mutant) were: probe 6: 5′-CTTTTCTGGCATTCGCGGTCACCTACCCG-3′ (wild type); probe 7: 5′-CTTTTCTGGCATTCGATTTCACCTACCCG-3′ (mutant). Mutated bases are underlined. DNA fragments were annealed by incubating sense and anti-sense DNA strands at 72°C for 10 minutes followed by slow cooling to room temperature and then labeled with 32P-γ-ATP using T4 polynucleotide kinase. Unincorporated nucleotides were removed using Micro Bio-spin P30 chromatography columns (Bio-Rad). DNA-protein binding reactions were performed in solution with 4% glycerol, 1 mmol/L MgCl2, 0.5 mmol/L ethylenediamine tetraacetic acid, 0.5 mmol/L dithiothreitol, 50 mmol/L NaCl, 10 mmol/L Tris-HCl, pH 7.5, and 0.05 mg/ml poly(dI-dC). Nuclear lysates were preincubated for 10 minutes, and then the labeled probes were added and incubated for an additional 20 minutes at room temperature. Competition analyses were performed in the presence of a 50-fold excess of unlabeled oligonucleotides. For the super shift assay, 2 μg of E2F-1 antibody (Santa Cruz Biotechnology) was included in the preincubation mixture. DNA-protein complexes were separated on a 6% DNA retardation gel (Invitrogen, Carlsbad, CA) in 0.5× Tris borate-ethylenediamine tetraacetic acid at 250 V for 1 hour. The gel was dried and exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) or to a PhosphorImager screen (GE Healthcare, Little Chalfont, Buckinghamshire, UK) for 4 hours and then scanned by phosphorimager. Controls included nuclear proteins from SAMe-treated KB cells, PC3 cells, and HeLa cells. The specific DNA-binding activity of NF-κB was measured by ELISA using the TransAM NF-κB p65 transcription factor assay kit (Active Motif North America, Carlsbad, CA). Nuclear proteins were incubated in 96-well plates with immobilized oligonucleotide containing a consensus binding site for the p65 subunit of NF-κB (5′-GGGACTTTCC-3′), which specifically binds the active form of NF-κB. A p65 antibody specific for the activated form was incubated with nuclear extract, and anti-IgG horseradish peroxidase-conjugated secondary antibody was used for visualization. The binding activity to NF-κB was detected and quantified by spectrophotometry at 450 nm with a reference wavelength of 655 nm. HeLa cells nuclear extract and lysis buffer without proteins were used as positive control and blank control, respectively. After treatment with SAMe for 30 minutes, cells were washed once with PBS (pH 7.4) and incubated with 1.2 ml of RIPA buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) with protease inhibitors (aprotinin, 30 μg/ml; leupeptin, 4 μg/ml; pepstatin, 2 μg/ml; and phenylmethyl sulfonyl fluoride, 10 μg/ml). The lysates were transferred to 1.7-ml Eppendorf tubes, and the plates were washed with 1.2 ml of RIPA buffer to ensure complete retrieval. Lysates were incubated for 30 minutes on ice and then centrifuged at 10,000 × g for 10 minutes at 4°C. After centrifugation, the protease inhibitor cocktail was immediately added to the supernatant, and protein concentration was determined by Bradford assay. Total proteins (70 μg) were mixed with electrophoresis sample buffer, boiled for 5 minutes, and separated on 14% Tris-glycine gels (Invitrogen). After electrophoresis, proteins were transferred to a PVDF membrane (American Pharmacia Biotech, Piscataway, NJ). Blots were probed with rabbit anti-human inhibitor κB-α (IκBα) and IκBβ polyclonal antibodies (Santa Cruz Biotechnology). α-Tubulin was used as an internal control. Goat anti-rabbit alkaline phosphatase-conjugated IgG was used as the secondary antibodies. Blots were incubated with Tropix CSPD chemiluminescent substrate and detected by the Tropix Western-Light and Western Star detection system (Bedford, MA). From the above study, GADD45β expression in Hep3B could not be induced by SAMe apparently as HepG2. Moreover, NF-κB-binding ability and activity failed to respond to SAMe administration. Based on the distinct difference of p53 status between HepG2 (p53 wild type) and Hep3B (p53-null), Hep3B cells were transiently transfected with 0.1 μg of pp53-EGFP (wild-type p53 fused to enhanced green fluorescent protein, GFP) (Clontech, Palo Alto, CA) by electroporation at parameter 80 μs/650 V. Mock transfection was included at the same time. Transfection efficiency was determined by counting the number of GFP-expressing cells per randomly chosen field of 100 cells 12 hours after infection. Then, promoter activity changes were investigated after SAMe treatment by the luciferase reporter assay. Transcriptional activity modifications were further explored by EMSA analyses, ELISA, and Western blot as mentioned above. Expression of GADD45β, as shown by Northern blot, was low in HepG2 cells and could be significantly induced by SAMe in a dose-dependent manner (Figure 1). There was approximately a fivefold increase in GADD45β mRNA with 0.5 mmol/L SAMe and an eightfold increase with 1.0 mmol/L SAMe. Although a lack of GADD45β expression was also observed in Hep3B as well as HepG2, induction by SAMe was barely observed in Hep3B by SAMe. Only a slight increase of GADD45β occurred at 0.5 mmol/L SAMe administration, and further escalation in SAMe dose led to little increase in the induction. Quantitative real-time PCR was used to further confirm the results from Northern blot. The standard curve formulas Y = 40.722 − 3.885X (r2 = 0.984) for GADD45β and Y = 43.128 − 4.248X (r2 = 0.993) for GAPDH were derived from the lines of the calibration curves. The mean ratio of GADD45β to GAPDH mRNA in untreated HepG2 cells was 0.0052. The mean ratios significantly increased to 0.0282 and 0.0525 for cells treated with 0.5 and 1.0 mmol/L SAMe, respectively (P < 0.05). Consistent with the results from Northern blot, Hep3B did not demonstrate apparent GADD45β induction. The mean ratio of GADD45β to GAPDH was 0.0097, and the mean ratios were kept stable in the range of 0.0104 to 0.0113 (P > 0.05). In our previous study, we identified several active promoter regions of GADD45β in HepG2 and CL-48.5Qiu W Zhou B Zou H Liu X Chu PG Lopez R Shih J Chung C Yen Y Hypermethylation of growth arrest DNA damage-inducible gene 45 beta promoter in human hepatocellular carcinoma.Am J Pathol. 2004; 165: 1689-1699Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Most of the active regions were located in GADD45β promoter fragment spanning −618 to −436. The promoter activity peak appeared at −470 with the deletion of as few as 50 bp from the 5′-end of −520. Therefore, a putative inhibitory region located between −520 and −470 was taken into consideration. By means of 5-Aza-dC treatment, methylation-specific PCR (MSP) and sequencing of sodium bisulfite-treated DNA, we confirmed the hypermethylation in the putative inhibitory region at −520/−470. As shown in Figure 2, although the promoter activity peak was detected at bp −470, deletion of as few as 34 bases from the 5′-end of this region led to a 24-fold decrease in promoter activity. One putative E2F-1-binding site (−452 to −444) was located in this area with high score in search of the TRANAFAC database. Meanwhile, the removal of 27 bases from −618 caused a significant decrease in promoter activity as well. One putative NF-κB-binding site (−602 to −593) was located in this deletion fragment with high score in the TRANAFAC database. Although further truncation from −592 and −547 could not influence promoter activity apparently, another two putative NF-κB-binding sites between −591 and −520 (−581 to −572 and −537 to −528) were also identified with a relatively low score in the database. Of interest, Hep3B shared the same pattern of proximal promoter activity as HepG2 despite the difference in p53 status. The overall activity level of Hep3B was relatively lower than that of HepG2 (Figure 2). Altogether, several active promoter regions and putative transcriptional factor-binding sites were identified based on luciferase report assay and consensus sequence. However, further evidence at the functional level is required to confirm the role of these regions in GADD45β regulation. From above, we confirmed the induction of GADD45β by SAMe in a dose-dependent manner, which represents a very useful model for regulation study. Therefore, to reveal functional evidence of putative promoter regions, the effects of SAMe on transcription factor-binding sites were focused on three putative NF-κB-binding sites (−618 to −470) and one putative E2F-1-binding site (−470 and −436), as determined using the luciferase reporter assay system. As shown in Figure 3A, luciferase activity increased approximately fourfold on the addition of SAMe in HepG2 transfected with the full-length reporter construct, which contained all three NF-κB-binding sites (−618 to +6). In the absence of SAMe, deletion of the first NF-κB-binding site (−591 to +6) led to a 70% decrease in promoter activity compared with the full-length construct. However, this construct retained the ability to be activated approximately twofold by SAMe treatment. The construct containing the third NF-κB-binding site (−547 to +6) could only be induced by SAMe approximately onefold. Meanwhile, there was not any response to SAMe for the E2F-1-binding site (−470 to −436) as well. In Figure 3B, the consistent failure to response to SAMe was observed, although even Hep3B had the same promoter activity profile, whereas the stimulator did not influence luciferase activity significantly. Although both NF-κB- and E2F-1-binding sites were identified in the proximal promoter region of GADD45β, only the NF-κB sites functioned positively to regulate transcription of GADD45β in response to SAMe treatment. We further validated the specificity of these sites in response to SAMe treatment by EMSA analysis using γ-32P-labeled oligonucleotide containing wild-type or mutant NF-κB and E2F-1 recognition motif and HepG2 nuclear lysates. Furthermore, anti-human E2F-1 antibody was used for the super shift assay to confirm binding specificity. As shown in Figure 4A, binding to the probe containing the first two NF-κB-recognition motifs (probe 1) in HepG2 nuclear lysates was substantially enhanced by SAMe in a dose-dependent manner. The addition of a 50-fold excess of unlabeled NF-κB oligonucleotide shifted the binding, indicating the specificity of the observed interaction. Binding to probe 2, in which the first NF-κB site was mutated, could not be readily detected. When the second NF-κB site was mutated (probe 3), no NF-κB-specific binding occurred, and the binding pattern was altered. No binding could be observed with mutation of both NF-κB-binding sites (probe 4). A probe containing only the third NF-κB recognition motif (probe 5) also yielded no binding activity. A probe containing the consensus NF-κB-binding sequence generated the same results as that obtained with probe 1 (data not shown). These results suggest
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