Cell Growth Inhibition and Gene Expression Induced by the Histone Deacetylase Inhibitor, Trichostatin A, on Human Hepatoma Cells

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Cell Growth Inhibition and Gene Expression Induced by the Histone Deacetylase Inhibitor, Trichostatin A, on Human Hepatoma Cells
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  Fax +41 61 306 12 34E-Mail karger@karger.chwww.karger.com Accessible online at:www.karger.com/ocl Laboratory Investigation  Oncology 2004;66:481–491 DOI: 10.1159/000079503 Cell Growth Inhibition and Gene Expression Induced by the Histone Deacetylase Inhibitor, Trichostatin A, on Human Hepatoma Cells Tetsuhiro Chiba Osamu Yokosuka Kenichi Fukai Hiroshige Kojima Motohisa Tada Makoto Arai Fumio Imazeki Hiromitsu Saisho Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba , Japan the potential of TSA to induce apoptosis. The microar-ray analysis revealed that 8 genes including collagen type 1, · 2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), integrin, · 7 (ITGA7), basigin (BSG), quiescin Q6 (QSCN6), superoxide dismutase 3, extra-cellular (SOD3), nerve growth factor receptor (NGFR), and p53-induced protein (PIG11) exhibited substantial induction (ratio 1 2.0) after TSA treatment in multiple cell lines. ChIP assay, in general, showed a good corre-lation between the expression level of mRNA and lev-els of acetylated histones in these upregulated genes. Conclusions: This study showed cell growth inhibition and the gene expression profile in hepatoma cell lines exposed to TSA. The alteration in levels of acetylated histones was closely associated with expression of spe-cific cancer-related genes in hepatoma cells. Copyright © 2004 S. Karger AG, Basel Introduction Hepatoma is one of the most common and important malignancies worldwide. Despite development in diag-nosis, treatment and prevention in hepatoma, advanced cases accompanied by multicentric carcinogenesis and portal vein tumor thrombus have so far had a poor prog-nosis [1–3] . It was suggested that genetic alterations, such as loss of heterozygosity and mutation of tumor suppres-  Key Words Trichostatin A · Histone acetylation · Hepatoma · cDNA microarray · Chromatin immunoprecipitation  Abstract Objective: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apopto-sis and differentiation in tumor cells. The effect of the HDAC inhibitor, trichostatin A (TSA), on hepatoma cells, however, has not been well studied. In this study, we ex-amined cell viability and gene expression profile in hep-atoma cell lines treated with TSA. Methods: To study cell growth inhibition and induction of apoptosis by TSA on human hepatoma cell lines including HuH7, Hep3B, HepG2, and PLC/PRF/5, cells were treated with TSA at various concentrations and analyzed by the 3-(4, 5-di-methyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively. Changes in gene expres-sion profile after exposure to TSA were assessed using a cDNA microarray consisting of 557 distinct cDNA of cancer-related genes. The levels of acetylated histones were examined by the chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 or H4 an-tibody. Results: The MTT assay demonstrated that TSA showed cell growth inhibition not only in a concentra-tion-dependent but also a time-dependent manner on all cell lines studied. The TUNEL assay also revealed Received: June 17, 2003 Accepted after revision: November 12, 2003 Oncology Osamu Yokosuka, MDDepartment of Medicine and Clinical OncologyGraduate School of Medicine, Chiba University1-8-1 Inohana, Chuo Ward, Chiba 260-8670 (Japan)Tel. +81 43 2262083, Fax +81 43 2262088, E-Mail yokosukao@faculty.chiba-u.jp © 2004 S. Karger AG, Basel0030–2414/04/0666–0481$21.00/0   Chiba/Yokosuka/Fukai/Kojima/Tada/Arai/Imazeki/Saisho Oncology 2004;66:481–491 482 sor genes, accumulate during multistep hepatocarcino-genesis [4, 5] . Recently, epigenetic alterations including histone deacetylation and DNA methylation in promoter areas were also hypothesized to play crucial roles in the development of hepatoma. Some studies reported that the combinatorial nature of histone amino-terminal mod-ifications, so-called ‘histone code’, contributes to a tran-scriptionally active chromatin status by interposition of protein modules, which may extend the information po-tential of the genetic code [6, 7] . The acetylation level in nucleosomal histones, which is controlled by interactions between histone acetyl transferase and histone deacety-lase (HDAC), regulates the chromatin structure and tran-scriptional activity [8, 9] . Chromatin fractions with ac-tively transcribed genes accompany the accumulation of acetylated histones, whereas silenced genes are associated with hypoacetylated histones [10, 11] . The HDAC inhibitor, trichostatin A (TSA) was ini-tially characterized as an anti-fungal drug and later found to inhibit HDAC activity strongly at nanomolar concen-trations [12] . The anti-proliferative effect of TSA was demonstrated in some malignancies [13, 14]; however, it has not been well studied in hepatoma cells. HDAC in-hibitors are estimated to cause apoptosis, cell cycle arrest and differentiation by inducing expression of several genes, such as p21  WAF1 in vitro [15, 16] , although only a small number of genes that altered expression in response to HDAC inhibitors have been identified. In the present study, we estimated the effect of TSA on cell growth and identified genes upregulated after TSA treatment on human hepatoma cells utilizing cDNA mi-croarray analysis. Furthermore, we investigated whether an increase in mRNA expression in these upregulated genes is actually accompanied by accumulation of acety-lated histones H3 and H4 using a chromatin immunopre-cipitation (ChIP) assay. Materials and Methods Cell Cultures and TSA Treatment The human hepatoma cell lines, HuH7, Hep3B, HepG2, PLC/PRF/5 were cultured in Dulbecco’s modified Eagle’s medium (In-vitrogen Life Technologies, Carlsbad, Calif., USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (In-vitrogen). HuH7, Hep3B and PLC/PRF/5 cells srcinating from hepatoma, and HepG2 cells were from hepatoblastoma. In contrast to Hep3B and PLC/PRF/5 cells, HuH7 and HepG2 cells lack inte-gration of hepatitis B virus sequences [17] . HepG2 cells contain the wild type of p53, although Hep3B cells are known to have deleted p53. HuH7 and PLC/PRF/5 cells have the mutant type of p53 [18] . Moreover, normal Rb is observed in PLC/PRF/5 cells, whereas that in other cell lines is functionally impaired [19] . The cells were in-cubated at 37 °   C in a humidified atmosphere of 5% CO  2 in air. TSA was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). The drug was dissolved in ethanol at a concentration of 100 mg/ml and was stored at –20 °   C. Cells were cultured at 2 ! 10  5 per milliliter in 10-cm dishes with 200 ng/ml of TSA for 24 h. Cell Growth Inhibition Cells were cultured in medium containing 0, 100, 200, 500, 1,000 and 2,000 ng/ml of TSA for 12, 24 or 36 h. Cell viability was examined using the 3-(4,5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) assay with duplicate samples as previously de-scribed [20] . Detection of Apoptotic Cells Quantification of apoptotic cells was performed by TUNEL as-say with duplicate samples according to the manufacturer’s instruc-tions (Takara Bio Inc., Otsu, Japan). Hepatoma cells treated with 0, 100, 200, 500, 1,000 and 2,000 ng/ml of TSA for 12, 24 or 36 h were fixed in 10% paraformaldehyde and treated with 0.3% H  2 O  2 for 30 min  . The cells were labeled with fluorescein isothiocyanate (FITC) using terminal deoxynucleotidyl transferase, and DNA free ends were stained with anti-FITC horseradish peroxidase and di-aminobenzidine. The proportion of TUNEL-positive cells was cal-culated by counting at least 500 cells randomly. cDNA Microarray Analysis Total RNA of cells including HuH7, Hep3B, HepG2 and PLC/PRF/5 with or without 200 ng/ml of TSA for 24 h was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. After isolation of polyA-RNA, cDNA derived from cells with or without TSA treatment was synthesized by amplifica-tion of 1 Ì g of polyA-RNA and labeled with Cy5-dUTP and Cy3-dUTP, respectively, using oligo dT(18) primer and reverse tran-scriptase. The labeled cDNA was hybridized to Intelligene Human cancer CHIP Ver. 2.1 (Takara Bio Inc.) spotted with the known 557 cancer-related genes. Before scanning, the slides were washed in 2  ! SSC/0.2% SDS 3 times and finally in 0.05  ! SSC. The hybrid-ized arrays were scanned using an Affymetrix 428 Array Scanner, and data images were analyzed using Biodiscovery Imagene Ver. 4.2. Signal intensities of DNA spots were corrected by housekeep-ing gene normalization. Semi-Quantitative RT-PCR RT was carried out using First strand superscript (Invitrogen) after pretreatment with amplification grade DNase I (Invitrogen). The primer sequence of each gene and PCR conditions are shown in table 1 . The PCR products were separated by electrophoresis on 3% agarose gel and visualized by UV light illumination using SYBR Green (Biowhittaker Molecular Applications, Rockland, Me., USA) staining. The intensity of the bands was quantified by using NIH Image 1.62 analysis software. The fold induction (intensity of PCR products with TSA treatment normalized by ß -actin expres-sion/intensity of untreated PCR products normalized by ß -actin expression) was calculated for each gene. ChIP Assay Formaldehyde was added to the cells with or without 200 ng/ml of TSA for 24 h to a final concentration of 1% to cross-link histones to DNA, and the cells were incubated at 37 °   C for 10 min. The me-dium was removed, and the cells were suspended in 1 ml of ice-cold   Cell Growth Inhibition and Gene Expression by TSA on Hepatoma Cells Oncology 2004;66:481–491 483PBS containing protease inhibitors. The cells were pelleted and re-suspended in 200 Ì l of SDS lysis buffer (1% SDS/10 m   M EDTA/50 m   M Tris-HCl, pH 8.1) and incubated on ice for 10 min. Lysates were sonicated to achieve the chromatin solution, and debris was removed by centrifugation at 12,000  g for 10 min at 4 °   C. The chro-matin solution was diluted 10-fold in TE buffer (50 m   M Tris-HCl/1 m   M EDTA, pH 8.1), and 80 Ì l of salmon sperm DNA/Protein A agarose slurry was added and incubated, rocking for 30 min at 4 °   C. Beads were pelleted by centrifugation, and supernatants were placed in new tubes with 5 Ì g of polyclonal rabbit anti-acetylated histone H3 or H4 antibody (Upstate Biotechnology Inc., Lake Placid, N.Y., USA) and incubated overnight at 4 °   C. Sixty micro-liters of salmon sperm DNA/Protein A agarose slurry was added, and samples were rocked for 1 h at 4 °   C. Protein A complexes were washed 5 times for 3 min each. The complexes were then eluted twice with 250 Ì l of elution buffer (1% SDS/0.1  M NaHCO  3 ) for 15 min at room temperature. Twenty microliters of 5  M NaCl was added to the eluates, and the samples were incubated at 65 °   C for 4 h to reverse histone-DNA cross-links. Then, 0.5  M EDTA, 1  M Tris-HCl and proteinase K were added to eluates, and incubated for 1 h at 45 °   C. Immunoprecipitated DNA was recovered by phe-nol/chloroform extraction and ethanol precipitation and analyzed by PCR. Specific primers from the promoter region of each gene and PCR condition are shown in table 2 . Quantification of acety-layed histones was also performed using NIH Image 1.62 analysis software. Table 1.  Primers used for RT-PCR and the ChIP assay Gene namePrimersBase pairsTempera-ture, °CCyclesRT-PCRCOL1A2Fw 5  -GACCTCCAGGTGTAAGCGGT-3  3325525Rv 5  -TTCAGGTTGGGCCCGGATAC-3  IGFBP2Fw 5  -TCTACAATGAGCAGCAGGAG-3  2605525Rv 5  -AAGCAAGAAGGAGCAGGTGT-3  ITGA7Fw 5  -AAGACCGACAGCAGTTCAAG-3  3615525Rv 5  -TACCCACTCTCATCTCACAG-3  BSGFw 5  -AATTTTATGAGGGCCACGGG-3  3085525Rv 5  -CGATCTTTATTGTGGCGGTG-3  QSCN6Fw 5  -CTCTTTAGCACCACATTCCT-3  5205525Rv 5  -GACAAAAGACCAGGCTCAGA-3  SOD3Fw 5  -CTAAGTGCCAGACCCAAGTT-3  3135525Rv 5  -AGTTAGGGGGCGTTGTAGTA-3  NGFRFw 5  -ATGAAGAAAAGCGGGCCAGT-3  5265530Rv 5  -AAGGGTTCCATCTCAGCTCA-3  PIG11Fw 5  -TACTGGGTGGCTTGGTTTAG-3  3505532Rv 5  -CTTGATTTGGGGTTGGGGAT-3  ß -actinFw 5  -ATCCTGCGTCTGGACCTGGCTGG-3  5205520Rv 5  -ACATGCCGGAGCCGTTGTCGACGA-3  ChIP assayCOL1A2Fw 5  -GGCTCAGGGTAGAACTGGTA-3   2316035Rv 5  -TCTAGACTAGACCGAGTCAC-3  IGFBP2Fw 5  -CTGTCACCCAAGGAATCTCTCT-3   2495035Rv 5  -GTCTTGGAGAGGTCAGATCAGC-3  ITGA7Fw 5  -CTTGGGTCCAGTCTCTTTCATC-3   2525235Rv 5  -GTCAGGATTCAAGGGAACAGAG-3  BSGFw 5  -GCAGGAAGGAAGAAATGCGC-3   2466035Rv 5  -TATAAAAAGCGGCGGAGGCG-3  §QSCN6Fw 5  -AGGCTCAAAGCTCTTACAGGTG-3  2505035Rv 5  -ATCCAGCTCTTCAGCCTACTCA-3  SOD3Fw 5  -GCTTTCTTGGACCTTAAACGAA-3   2515040Rv 5  -CACCTCTGCATTCTGTTGGTAG-3  NGFRFw 5  -GTACATATGCGCGTTTGAATGT-3   2525040Rv 5  -GACATTCAAGGTGGAGTCCATT-3  PIG11Fw 5  -TCCAGAGCCTGCTCTATTAACC-3  2485040Rv 5  -CCCTGTGCTCTCCTTAGAGAAA-3  ß -actinFw 5  -CTGCGCATAGCAGACATACAA-3   3525235Rv 5  -CTGGGCTTGAGAGGTAGAGTG-3    Chiba/Yokosuka/Fukai/Kojima/Tada/Arai/Imazeki/Saisho Oncology 2004;66:481–491 484  Results Cell Growth Inhibition of TSA on Hepatoma Cells Hepatoma cells exposed to TSA showed gradual mor-phological changes such as cytoplasmic elongation and loss of cell-to-cell contact. These morphological changes appeared to be concentration- and time-dependent. The MTT assay revealed TSA inhibited the growth of all hep-atoma cell lines studied, not only in a concentration-de-pendent but also in a time-dependent manner (fig. 1 ). Cell viability after 24 h treatment with 200 ng/ml of TSA on HuH7, Hep3B, HepG2, and PLC/PRF/5 cells was 74.3%, 90.8%, 76.0%, and 72.0%, respectively. Concerning p53 function, the viability of HepG2 cells with intact p53 showed little difference compared to other cell lines with impaired p53 function.  Apoptotic Cell Death by TSA Treatment TUNEL-positive nuclei were also detected in the 4 hepatoma cell lines; their number was concentration- and time-dependent (fig. 2 ). The percentage of apoptotic cells after 24 h of treatment with 200 ng/ml of TSA on HuH7, Hep3B, HepG2, and PLC/PRF/5 cells was 16.8, 13.0, 18.5, and 26.5, respectively. Gene Expression Profile Altered after the Exposure of TSA The cDNA microarray analysis demonstrated altered gene expression profiles after exposure to TSA. The num-ber of upregulated genes (ratio 1 2.0) in HuH7, Hep3B, HepG2, and PLC/PRF/5 cells is 24 (4.3%), 16 (2.9%), 13 (2.3%), and 11 (2.0%), respectively ( table 2 ). These up-regulated genes were classified according to function by referring to the literature or using available websites and were categorized into 7 groups as follows: apoptosis/cell cycle, cell growth and maintenance, cellular communica-tion/signal transduction, cell structure, metabolism, tran-scription and others. Many of the altered genes were in the apoptosis/cell cycle group; the number of apoptosis/cell cycle genes in HuH7, Hep3B, HepG2, and PLC/PRF/5 cells is 7, 7, 3 and 3, respectively. The expression of 8 genes, including collagen type 1  · 2 (COL1A2); insu-lin-like growth factor binding protein 2 (IGFBP2); integ-rin, · 7 (ITGA7); basigin (BSG); quiescin Q6 (QSCN6); superoxide dismutase 3, extracellular (SOD3); nerve growth factor receptor (NGFR), and p53-induced protein (PIG11), were increased (ratio 1 2.0) in at least 2 cell lines. Fig. 1. Cell growth inhibition by TSA. Four hepatoma cells including HuH7, Hep3B, HepG2, and PLC/PRF/5 were exposed to various concentrations of TSA for 12, 24 or 36 h. TSA shows cell concentration and time-dependent growth inhibition of allthe hepatoma cell lines studied. a HuH7.  b Hep3B. c HepG2. d PLC/PRF/5.   Cell Growth Inhibition and Gene Expression by TSA on Hepatoma Cells Oncology 2004;66:481–491 485 Accession a Gene name Symbol Fold-induction HuH7Hep3BHepG2PLC/PRF/5 micro-array b RT-PCR c micro-array RT-PCR micro-array RT-PCR micro-array RT-PCR  Apoptosis/cell cycle  d M14764 e Nerve growth factor receptorNGFR64.77  ND 27.97  ND ND3.64ND AF035752 Caveolin 2 CAV2   2.98   0.50   1.01 0.55 AF010315p53-induced protein  PIG11   2.88 2.17   2.84 3.06   3.13 1.42 ND U66879 BCL2-antagonist of cell death BAD   2.83   1.94   1.18 0.61 U97276Quiescin Q6  QSCN6   2.29 1.89   3.84 2.06   2.33 1.49 1.931.02 U60521 Caspase 9, apoptosis-related cysteineprotease CASP9   2.27   1.09   0.80 1.04X02812Transforming growth factor, beta 1 TGFB1   2.08   1.36   0.72 0.88AL023282Tissue inhibitor of metalloproteinase 3 TIMP3   1.85   1.30   1.54 2.44AF041248Cyclin-dependent kinase inhibitor 2C CDKN2C   1.76   2.14   1.72 1.24X61587Ras homolog gene family, member G ARHG   1.58   1.45   1.13 2.02U82938CD27-binding protein CD27BP   1.46   2.73   1.82 0.94X86779Fas-activated serine/threonine kinase FASTK    0.77   2.11   1.62 1.21J00117Chorionic gonadotropin, betapolypeptide CGB 6.20U43142Vascular endothelial growth factor C VEGFC   2.09 Cell growth and maintenance  AF032108Integrin, alpha 7ITGA710.66  4.58   1.45 1.86   3.39 2.48 1.78  X16302 Insulin-like growth factor bindingprotein 2  IGFBP2   3.59 2.06 25.86  6.80 1.552.852.90 X78947 Connective tissue growth factor CTGF   2.90   0.51 0.41AL110197Tissue inhibitor of metalloproteinase 2 TIMP2   2.80   0.77   0.99 0.82 Table 2.  Genes upregulated by TSA treatment   Fig. 2. Induction of apoptosis by TSA. The 4 hepatoma cell lines were treated with sev-eral concentrations of TSA for 12, 24, or36 h. Concentration- and time-dependent induction of apoptosis was observed in these cell lines. a HuH7. b Hep3B. c HepG2. d PLC/PRF/5.
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