Cell Growth Inhibition and Gene Expression Induced by ...

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Laboratory Investigation

Oncology

Oncology 2004;66:481–491 DOI: 10.1159/000079503

Received: June 17, 2003 Accepted after revision: November 12, 2003

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

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, apoptosis 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 examined cell viability and gene expression profile in hepatoma 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-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively. Changes in gene expression 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 antibody. Results: The MTT assay demonstrated that TSA showed cell growth inhibition not only in a concentration-dependent but also a time-dependent manner on all cell lines studied. The TUNEL assay also revealed

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the potential of TSA to induce apoptosis. The microarray 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, extracellular (SOD3), nerve growth factor receptor (NGFR), and p53-induced protein (PIG11) exhibited substantial induction (ratio 12.0) after TSA treatment in multiple cell lines. ChIP assay, in general, showed a good correlation between the expression level of mRNA and levels 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 specific 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 diagnosis, treatment and prevention in hepatoma, advanced cases accompanied by multicentric carcinogenesis and portal vein tumor thrombus have so far had a poor prognosis [1–3]. It was suggested that genetic alterations, such as loss of heterozygosity and mutation of tumor suppres-

Osamu Yokosuka, MD Department of Medicine and Clinical Oncology Graduate School of Medicine, Chiba University 1-8-1 Inohana, Chuo Ward, Chiba 260-8670 (Japan) Tel. +81 43 2262083, Fax +81 43 2262088, E-Mail [email protected]

sor genes, accumulate during multistep hepatocarcinogenesis [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 modiﬁcations, so-called ‘histone code’, contributes to a transcriptionally active chromatin status by interposition of protein modules, which may extend the information potential of the genetic code [6, 7]. The acetylation level in nucleosomal histones, which is controlled by interactions between histone acetyl transferase and histone deacetylase (HDAC), regulates the chromatin structure and transcriptional activity [8, 9]. Chromatin fractions with actively 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 initially characterized as an anti-fungal drug and later found to inhibit HDAC activity strongly at nanomolar concentrations [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 inhibitors are estimated to cause apoptosis, cell cycle arrest and differentiation by inducing expression of several genes, such as p21WAF1 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 microarray analysis. Furthermore, we investigated whether an increase in mRNA expression in these upregulated genes is actually accompanied by accumulation of acetylated histones H3 and H4 using a chromatin immunoprecipitation (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 (Invitrogen Life Technologies, Carlsbad, Calif., USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen). HuH7, Hep3B and PLC/PRF/5 cells originating from hepatoma, and HepG2 cells were from hepatoblastoma. In contrast to Hep3B and PLC/PRF/5 cells, HuH7 and HepG2 cells lack integration 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-

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cubated at 37 ° C in a humidiﬁed atmosphere of 5% CO2 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 ! 105 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 described [20]. Detection of Apoptotic Cells Quantiﬁcation of apoptotic cells was performed by TUNEL assay with duplicate samples according to the manufacturer’s instructions (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% H2O2 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 diaminobenzidine. The proportion of TUNEL-positive cells was calculated 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 ampliﬁcation of 1 Ìg of polyA-RNA and labeled with Cy5-dUTP and Cy3dUTP, respectively, using oligo dT(18) primer and reverse transcriptase. 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 hybridized 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 housekeeping 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 expression/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 medium was removed, and the cells were suspended in 1 ml of ice-cold

Chiba/Yokosuka/Fukai/Kojima/Tada/ Arai/Imazeki/Saisho

Table 1. Primers used for RT-PCR and

the ChIP assay

Gene name RT-PCR COL1A2 IGFBP2 ITGA7 BSG QSCN6 SOD3 NGFR PIG11 ß-actin

Primers

Base Tempera- Cycles pairs ture, °C

Fw 5-GACCTCCAGGTGTAAGCGGT-3 Rv 5-TTCAGGTTGGGCCCGGATAC-3 Fw 5-TCTACAATGAGCAGCAGGAG-3 Rv 5-AAGCAAGAAGGAGCAGGTGT-3 Fw 5-AAGACCGACAGCAGTTCAAG-3 Rv 5-TACCCACTCTCATCTCACAG-3 Fw 5-AATTTTATGAGGGCCACGGG-3 Rv 5-CGATCTTTATTGTGGCGGTG-3 Fw 5-CTCTTTAGCACCACATTCCT-3 Rv 5-GACAAAAGACCAGGCTCAGA-3 Fw 5-CTAAGTGCCAGACCCAAGTT-3 Rv 5-AGTTAGGGGGCGTTGTAGTA-3 Fw 5-ATGAAGAAAAGCGGGCCAGT-3 Rv 5-AAGGGTTCCATCTCAGCTCA-3 Fw 5-TACTGGGTGGCTTGGTTTAG-3 Rv 5-CTTGATTTGGGGTTGGGGAT-3 Fw 5-ATCCTGCGTCTGGACCTGGCTGG-3 Rv 5-ACATGCCGGAGCCGTTGTCGACGA-3

332

55

25

260

55

25

361

55

25

308

55

25

520

55

25

313

55

25

526

55

30

350

55

32

520

55

20

231

60

35

249

50

35

252

52

35

246

60

35

250

50

35

251

50

40

252

50

40

248

50

40

352

52

35

ChIP assay COL1A2 Fw 5-GGCTCAGGGTAGAACTGGTA-3 Rv 5-TCTAGACTAGACCGAGTCAC-3 IGFBP2 Fw 5-CTGTCACCCAAGGAATCTCTCT-3 Rv 5-GTCTTGGAGAGGTCAGATCAGC-3 ITGA7 Fw 5-CTTGGGTCCAGTCTCTTTCATC-3 Rv 5-GTCAGGATTCAAGGGAACAGAG-3 BSG Fw 5-GCAGGAAGGAAGAAATGCGC-3 Rv 5-TATAAAAAGCGGCGGAGGCG-3§ QSCN6 Fw 5-AGGCTCAAAGCTCTTACAGGTG-3 Rv 5-ATCCAGCTCTTCAGCCTACTCA-3 SOD3 Fw 5-GCTTTCTTGGACCTTAAACGAA-3 Rv 5-CACCTCTGCATTCTGTTGGTAG-3 NGFR Fw 5-GTACATATGCGCGTTTGAATGT-3 Rv 5-GACATTCAAGGTGGAGTCCATT-3 PIG11 Fw 5-TCCAGAGCCTGCTCTATTAACC-3 Rv 5-CCCTGTGCTCTCCTTAGAGAAA-3 Fw 5-CTGCGCATAGCAGACATACAA-3 ß-actin Rv 5-CTGGGCTTGAGAGGTAGAGTG-3

PBS containing protease inhibitors. The cells were pelleted and resuspended in 200 Ìl of SDS lysis buffer (1% SDS/10 mM EDTA/ 50 mM 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 chromatin solution was diluted 10-fold in TE buffer (50 mM Tris-HCl/ 1 mM EDTA, pH 8.1), and 80 Ìl 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 Ìg 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 microliters 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 Ìl of elution buffer (1% SDS/0.1 M NaHCO3) 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 phenol/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 acetylayed histones was also performed using NIH Image 1.62 analysis software.

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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 all the hepatoma cell lines studied. a HuH7. b Hep3B. c HepG2. d PLC/PRF/5.

Results

Cell Growth Inhibition of TSA on Hepatoma Cells Hepatoma cells exposed to TSA showed gradual morphological 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 hepatoma cell lines studied, not only in a concentration-dependent 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.

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Gene Expression Proﬁle Altered after the Exposure of TSA The cDNA microarray analysis demonstrated altered gene expression proﬁles after exposure to TSA. The number of upregulated genes (ratio 12.0) 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 upregulated 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 communication/signal transduction, cell structure, metabolism, transcription 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); insulin-like growth factor binding protein 2 (IGFBP2); integrin, ·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 12.0) in at least 2 cell lines.

Chiba/Yokosuka/Fukai/Kojima/Tada/ Arai/Imazeki/Saisho

Fig. 2. Induction of apoptosis by TSA. The 4 hepatoma cell lines were treated with several concentrations of TSA for 12, 24, or 36 h. Concentration- and time-dependent induction of apoptosis was observed in these cell lines. a HuH7. b Hep3B. c HepG2. d PLC/PRF/5.

Table 2. Genes upregulated by TSA treatment Accessiona

Gene name

Symbol

Fold-induction HuH7

Apoptosis/cell cycled M14764e Nerve growth factor receptor AF035752 Caveolin 2 AF010315 p53-induced protein U66879 BCL2-antagonist of cell death U97276 Quiescin Q6 U60521 Caspase 9, apoptosis-related cysteine protease X02812 Transforming growth factor, beta 1 AL023282 Tissue inhibitor of metalloproteinase 3 AF041248 Cyclin-dependent kinase inhibitor 2C X61587 Ras homolog gene family, member G U82938 CD27-binding protein X86779 Fas-activated serine/threonine kinase J00117 Chorionic gonadotropin, beta polypeptide U43142 Vascular endothelial growth factor C Cell growth and maintenance AF032108 Integrin, alpha 7 X16302 Insulin-like growth factor binding protein 2 X78947 Connective tissue growth factor AL110197 Tissue inhibitor of metalloproteinase 2

Cell Growth Inhibition and Gene Expression by TSA on Hepatoma Cells

NGFR CAV2 PIG11 BAD QSCN6 CASP9 TGFB1 TIMP3 CDKN2C ARHG CD27BP FASTK

IGFBP2 CTGF TIMP2

HepG2

microarrayb

RTPCRc

microarray

RTPCR

64.77 2.98 2.88 2.83 2.29

ND

27.97 0.50 2.84 1.94 3.84

ND

2.17 1.89

2.27 2.08 1.85 1.76 1.58 1.46 0.77

CGB VEGFC

ITGA7

Hep3B

3.06 2.06

1.09 1.36 1.30 2.14 1.45 2.73 2.11

microarray

1.01 3.13 1.18 2.33

PLC/PRF/5 RTPCR

microarray

RTPCR

ND

3.64 0.55

ND

1.42 1.49

0.80 0.72 1.54 1.72 1.13 1.82 1.62

ND 0.61 1.93

1.02

1.04 0.88 2.44 1.24 2.02 0.94 1.21

6.20 2.09

10.66

4.58

1.45

1.86

3.59 2.90 2.80

2.06

25.86 0.51 0.77

6.80

Oncology 2004;66:481–491

3.39

2.48 1.55

0.99

1.78 2.85 0.41 0.82

2.90

485

Table 2 (continued) Accessiona

Gene name

Symbol

Fold-induction HuH7 microarrayb

M59911 L20688 Z68228 X03168 M98399 L34657 NM001718

Integrin, alpha 3 Rho-GDP dissociation inhibitor beta Junction plakoglobin Vitronectin CD36 antigen Platelet/endothelial cell adhesion molecule Bone morphogenetic protein 6

ITGA3 ARHGDIB JUP VTN CD36

1.84 1.79 1.43 0.69

10.62 7.91 2.68 2.31 2.05

COL1A2 KRT19 KRT14 MMP19 MMP11 KRT7 MMP13 COL11A2 ANK1 LUM FBN2

2.17 2.13 1.61 1.29 1.15 0.64

Metabolism U10116 AF064594 Y00486

Superoxide dismutase 3, extracellular Phospholipase A2, group VI Adenine phosphoribosyltransferase

SOD3 PLA2G6 APRT

3.16 1.92 0.75

AJ001902 a b c d

e

RTPCR

3.17 0.90 1.12 8.73

1.50

0.78 2.57

microarray

RTPCR

0.80 12.15 1.32 2.26 1.37

1.51

0.42

Collagen type I, alpha 2 Keratin 19 Keratin 14 Matrix metalloproteinase 19 Matrix metalloproteinase 11 Keratin 7 Matrix metalloproteinase 13 Collagen type XI, alpha 2 Ankyrin 1, erythrocytic Lumican Fibrillin 2

Glutaredoxin Major histocompatibility complex, class I, A Thyroid hormone receptor interactor 6

microarray

PLC/PRF/5 microarray 1.09 1.86 2.14 0.53

3.90 0.89

2.14

1.00

0.90

1.95 1.32

RTPCR

2.02

1.27 1.88 0.76

0.97

2.07

Cell structure J03464 Y00503 J00124 U38320 X57766 AJ238246 X75308 AL031228 X16609 U21128 U03272

Others AF069668 D32129

RTPCRc

HepG2

PECAM BMP6

Cellular communication/signal transduction M38690 CD9 antigen CD9 U66406 Ephrin-B3 EFNB3 U43842 Bone morphologenetic protein 4 BMP4 X64364 Basigin BSG M60278 Diphteria toxin receptor DTR X13444 CD8 antigen, beta polypeptide 1 CD8B1 J03278 Platelet-derived growth factor receptor, beta polypeptide PDGFRB

Transcription V01512 V-fos FBJ murine osteosarcoma viral oncogene homolog L49169 FBJ murine osteosarcoma viral oncogene homolog B X51345 Jun B proto-oncogene

Hep3B

1.90

8.70 1.19 0.95 0.87 2.16 0.51

1.67

2.39

69.69 1.77 2.25

7.91

ND 1.79 2.79 0.86 0.49 2.24

0.62 239.29 8.26 3.65 0.95 0.25

0.69 0.21

1.66

ND 1.30 1.48 2.01

5.00 2.82

1.29

1.36

2.41 1.47

0.87

FOS

76.43

FOSB JUNB

29.31 2.63

0.62

1.73

0.68

GLRX

4.28

1.35

0.78

0.86

HLA-A TRIP6

2.06 0.15

1.90 0.85

1.83 1.15

1.13 5.47

GenBank accession number. Blank entries indicate that data points did not pass the microarray spot of housekeeping genes during data analysis. ND, not determined. Gene functions were summarized from literature sources or according to LocusLink in the National Center for Biotechnology Information (www.ncbi.nlm. nih.gov/LocusLink). Upregulated (ratio >2.0) genes at least in 2 cell lines are shown in bold type.

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Table 3. Genes downregulated (ratio <0.5) by TSA treatment in at least 2 cell lines based on cDNA microarray Accessiona

Gene name

Symbol

Fold inductionb HuH7

Hep3B

HepG2

PLC/PRF/5

0.37 0.52

Apoptosis/cell cycle U58334 Tumor protein p53-binding protein 2 Mitogen-activated protein kinase 6 X80692 Interleukin 8 M17017 Transforming growth factor, beta 2 M19154 Breast cancer 2, early onset X95152 Mouse double minute 2, human homolog of; p53-binding protein M92424 NM001165 Apoptosis inhibitor 2

TP53BP2 MAPK6 IL8 TGFB2 BRCA2 MDM2 API2

0.17 0.18 0.19 0.32 0.44

0.13 0.26 0.11 0.11 0.28 0.14 0.20

0.48 0.26 0.50

0.76 0.75 0.18 0.38 0.44 0.59 0.40

Cell growth and maintenance X68742 Integrin, alpha 1 Desmoplakin (DPI, DPII) AL031058 Topoisomerase (DNA) I M60706 Integrin, alpha V M14648 PTK2 protein tyrosine kinase 2 L13616 Translocated_promoter_region_(to_activated_MET_oncogene) X66397 X00588 Epidermal growth factor receptor

ITGA1 DSP TOP1 ITGAV PTK2 TPR EGFR

0.11 0.22 0.23 0.27 0.29 0.29 0.29

0.26 0.22 0.23 0.19 0.23 0.24 0.33

0.29 0.29 0.29 0.69 0.69 0.49 0.44

0.63 1.22 0.73 1.10 1.18 1.17 1.04

Cellular communication/signal transduction D86962 Growth factor receptor-bound protein 10 A disintegrin and metalloproteinase domain 9 (meltrin gamma) U41766 Carcinoembryonic antigen-related cell adhesion molecule 1 X16354 U43195 Rho-associated, coiled-coil containing protein kinase 1

GRB10 ADAM9 CEACAM1 ROCK1

0.21 0.24 0.27 0.28

0.37 0.24 0.27 0.28

0.34 0.47

0.77 0.74 0.40 0.82

Cell structure NM002291 M55210 NM004010

Laminin, beta 1 Laminin, gamma 1 (formerly LAMB2) Dystrophin (muscular dystrophy, Duchenne and Becker types)

LAMB1 LAMC1 DMD

0.20 0.25 0.28

0.22 0.26 0.28

0.42 0.55 0.71

0.82 0.89

Metabolism AF027156

Mannosidase, alpha, class 1A, member 2

MAN1A2

0.69

0.14

0.38

1.17

Others L22009 X84740

Heterogeneous nuclear ribonucleoprotein H1 (H) Ligase III, DNA, ATP-dependent

HNRPH1 DIG3

0.21 0.21

0.24 0.27

0.89 0.56

0.96 1.13

a b

0.39

GenBank accession number. Blank entries indicate that data points did not pass the microarray spot of housekeeping genes during data analysis.

On the other hand, the number of downregulated genes (ratio ! 0.5) in HuH7, Hep3B, HepG2, and PLC/PRF/5 cells is 49 (8.8%), 35 (6.3%), 26 (4.7%), and 15 (2.7%), respectively. The number of genes summarized in apoptosis/cell cycle in HuH7, Hep3B, HepG2, and PLC/ PRF/5 cells is 11, 11, 5 and 6, respectively. Twenty-ﬁve genes showed decreased (ratio !0.5) expression in at least 2 cell lines (table 3).

Altered mRNA Expression Based on SemiQuantitative RT-PCR We also carried out semi-quantitative RT-PCR (ﬁg. 3a) on the 8 genes showing upregulation (ratio 12.0) in multiple cells based on cDNA microarray to validate the results of the microarray analysis. Eight genes, including COL1A2, IGFBP2, ITGA7, BSG, QSCN6, SOD3, NGFR, and PIG11, showed upregulation by TSA treatment in multiple cell lines as detected with microarray analysis. COL1A2 in PLC/PRF/5 cells showed no expres-

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sion before or after TSA treatment. NGFR also showed no basal expression; however, expression was observed after TSA treatment in 4 hepatoma cells. PIG11 demonstrated increased expression levels by TSA in HuH7, Hep3B and HepG2 cells but no expression before or after TSA treatment in PLC/PRF/5 cells. Northern blot analysis (data not shown) showed results comparable to those of semi-quantitative RT-PCR for 6 genes including COL1A2, IGFBP2, ITGA7, BSG, QSCN6 and SOD3. However, NGFR and PIG 11 could not be detected, probably due to the low expression level of these genes by Northern blot. Association between Expression Level of mRNA and Histone Acetylation The ChIP assay using anti-acetylated histone H3 (ﬁg. 3b) or H4 antibody (ﬁg. 3c) showed similar gene patterns, although the level of acetylated histone H4 in COL1A2 in HepG2 cells was higher than that of acetylated histone H3. Increased levels of acetylated histones in the promoter region after TSA treatment almost correlated with the transcriptional activities based on RT-PCR in the 8 genes mentioned above (tables 2, 4). However, COL1A2 and SOD3 in PLC/PRF/5 cells showed a discrepancy between mRNA expression and histone acetylation (tables 2, 4).

Discussion

The MTT assay in the present study revealed that TSA displayed concentration-dependent and time-dependent cell growth inhibition of 4 hepatoma cell lines including HuH7, Hep3B, HepG2 and PLC/PRF/5. The observed cell growth inhibition by TSA in HepG2 cells with intact p53 was almost similar to that of other cell lines with impaired p53 function, which might indicate that TSA acts

Fig. 3. a Semi-quantitative RT-PCR in the following 8 upregulated genes: COL1A2, IGFBP2, ITGA7, BSG, QSCN6, SOD, NGFR, and PIG11. These genes show increased expression level as detected with microarray analysis. b, c Histone acetylation assessed by the ChIP assay using anti-acetylated histone H3 and H4 antibody in 8 genes upregulated in multiple cell lines after TSA treatment. Increased levels of acetylation on histones H3 were almost proportional to the mRNA expressions except for COL1A2 and SOD3 in PLC/PRF/5 cells (b). The levels of acetylated histones H4 in these genes were similar to those of acetylated histones H3 except for COL1A2 in HepG2 cells (c).

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Table 4. Fold induction of genes associated with acetylated histones

H3 and H4 by ChIP analysis Gene name COL1A2 IGFBP2 ITGA7 BSG QSCN6 SOD3 NGFR PIG11

Ac-H3 Ac-H4 Ac-H3 Ac-H4 Ac-H3 Ac-H4 Ac-H3 Ac-H4 Ac-H3 Ac-H4 Ac-H3 Ac-H4 Ac-H3 Ac-H4 Ac-H3 Ac-H4

HuH7

Hep3B HepG2 PLC/PRF/5

3.06 2.70 2.38 1.68 3.79 4.10 2.36 2.40 1.88 2.03 ND 3.23 ND 9.20 1.75 1.60

3.67 3.11 2.07 2.20 1.16 1.06 1.68 1.54 2.54 1.90 3.59 4.11 3.06 3.94 3.22 3.88

1.10 1.02 4.40 4.59 2.80 3.48 3.03 2.05 1.37 1.35 1.18 1.54 1.23 ND 2.10 2.59

2.19 2.08 2.29 2.72 1.09 1.11 1.07 1.66 0.58 0.88 2.85 2.20 0.96 ND 0.48 ND

Ac-H3 = Actylated histone H3; Ac-H4 = acetylated histone H4; ND = not determined.

independently of p53, as reported in breast and lung cancer cells [16, 17]. Our TUNEL assay results also showed that hepatoma cell growth inhibition by TSA was in part mediated by induction of apoptosis. The anti-proliferative effect and induction of apoptotic programs by TSA on several hepatoma cells such as Hep1B and MH1C1 cells was reported [23]. Taken together, HDAC inhibitors appear to possess potential as new therapeutic agents against hepatoma. It was reported that HDAC inhibitors enhance the expression of p21WAF1, p27KIP1 and p57, on the other hand, they decrease the expression of p53, c-myc, and IL-8 [24– 26]. Altered expression of a small number of genes in response to TSA has been reported in hepatoma cells. Recently, cDNA microarray has been utilized to screen for genes upregulated by DNA methyltransferase inhibiton or HDAC inhibition in carcinoma cell lines [27, 28]. In this study, we performed the screening of 557 selected cancer-related genes by TSA treatment using the cDNA microarray technique on multiple hepatoma cell lines. It was reported that TSA selectively affects gene expression and modulates the expression in approximately 2–5% of genes expressed in human lymphoid cells using differential display [29]. Based on our microarray analysis, however, the percentages of upregulated (ratio 12.0) or downregulated (ratio !0.5) genes in HuH7, Hep3B, HepG2,

Cell Growth Inhibition and Gene Expression by TSA on Hepatoma Cells

and PLC/PRF/5 cells are 13.1, 9.2, 7.0 and 4.7%, respectively. These results might depend on the characteristics of our microarray, which was spotted with the known 557 cancer-related genes. We performed microarray analysis of hepatoma cells treated with or without 200 ng/ml of TSA for 24 h, but the possibility also exists that different doses or treatment durations of TSA affect the gene expression proﬁle. It was reported that the anti-tumor effect of HDAC inhibitors is preferentially associated with altered expression of apoptosis-regulating and cell cycleregulating genes [30] and that TSA induced the increased expression of p21WAF1, a well-known factor regulating the cell cycle, on hepatoma cells [31]. Concerning apoptosis induction by TSA, upregulation of pro-apoptotic bax and downregulation of anti-apoptotic bcl-2 were also reported [32]. Our gene expression proﬁle based on microarray revealed that TSA actually affected the expression of comparatively many genes implicated in apoptosis and/or cell cycle. COL1A2, IGFBP2, ITGA7, BSG, QSCN6, SOD3, NGFR and PIG11 were upregulated (ratio 1 2.0) in multiple hepatoma cells studied. The results of Northern blot analysis and RT-PCR conﬁrmed the data of the microarray analysis. cDNA microarray appears to be a reliable technique now available to analyze the expression of numerous genes although slight differences in detection sensitivity might exist between microarray and conventional methods, such as Northern blot analysis and RTPCR. The present ChIP analysis showed that these genes generally displayed increased transcriptional activities accompanied with accumulation of acetylated histones H3 and H4 in promoter regions after TSA treatment. However, the possibility also exists that upregulation of other genes in our expression proﬁle was not the result of direct accumulation of acetylated histones but was induced by activation of upstream genes. The disproportion between mRNA expression and increased levels of histones such as COL1A2 and SOD3 in PLC/PRF/5 cells might be attributed to different acetylation patterns throughout the promoter regions [33]. Histone H3 and H4 are components of core histones, and N-terminal tails of those are modiﬁed by acetylation. Our results of ChIP analysis generally showed similar gene patterns in acetylated histones H3 and H4, suggesting that histones H3 and H4 contribute to transcriptional regulation equally and cooperatively. Our current study has revealed that HDAC inhibitor TSA could suppress cell growth and cause apoptosis in hepatoma cells. The analysis of gene expression after TSA

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treatment revealed the upregulation of genes involved in cell growth, cell cycle and apoptosis through acetylation of histones H3 and H4. Although no association between these genes and progression of hepatoma has been reported, some might be candidate tumor suppressor genes. For example, IGFBP2 is a member of the IGFBPs, which regulate insulin-like growth factor associated with growth control and carcinogenesis [34]. QSCN6 is known as negative regulator of cell proliferation in human embryo lung ﬁbroblasts [35]. NGFR was also reported be associated with apoptosis and differentiation in SH-SY5Y neuroblastoma cells [36]. Thus, TSA might cause cell growth inhibition through upregulation of such candidate tumor suppressor genes, although whether these genes actually cause cell growth inhibition needs to be conﬁrmed in the future. In addition, downregulated genes with oncogenic potential were detected by cDNA microarray (table 3). The downregulation of these genes would likely not be due to the direct changes of histone acetylation in the promoter region by TSA [10, 11], but rather would result from a downstream effect of the regulation of upstream genes. This downregulation of the oncogenic genes might be another reason for apoptosis induction and cell growth inhibition by TSA treatment. Aberrant methylation of CpG islands near regulatory regions of genes is also one of the crucial epigenetic alterations in human cancers and is associated with gene silencing. In our study, all the genes except PIG11 and SOD3 possess 5 CpG islands, and COL1A2 in HepG2 and PLC/PRF/5, and IGFBP2 in HepG2 showed dense methylation (data not shown). It was reported that methyl-CpG binding proteins such as methyl-CpG binding

protein 2 (MeCP2) and methylated-DNA binding domain 3 (MBD3) including HDACs and transcriptional corepressors are involved in the silencing process [37, 38]. Some studies showed that TSA could partially relieve the transcriptional repression mediated by aberrant DNA methylation at least in several genes, although the induction of gene expression is comparatively low [39]. Based on the present cDNA microarray and PCR analysis, TSA could induce a low level of gene expression for COL1A2 in HepG2 and PLC/PRF/5 cells and IGFBP2 in HepG2 cells. Recently, it was reported that the combination of TSA and DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (5azaCdR), synergistically enhanced the induction of some tumor suppressor genes and strongly induced apoptosis [40]. The synergistic effect induced by an HDAC inhibitor and a DNA methyltransferase inhibitor appears to be a promising mechanism to consider for clinical applications. In conclusion, we demonstrated the potential of an HDAC inhibitor as an anti-cancer agent in hepatoma which was supported by the gene expression proﬁle after TSA treatment using cDNA microarray. Further investigation of the modiﬁcations of histone tails is needed in order to clarify the mechanisms underlying hepatocarcinogenesis and explore new therapeutic approaches.

Acknowledgement The authors thank Dr. Masakatsu Yamashita (Department of Molecular Immunology, Graduate School of Medicine, Chiba University) for helpful suggestions with the ChIP assay.

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