Sequential gene expression changes in cancer cell lines after treatment with the demethylation agent...

Sequential Gene Expression Changes in Cancer CellLines after Treatment with the Demethylation Agent5-Aza-2�-Deoxycytidine

Makoto Arai, M.D.1,2

Osamu Yokosuka, M.D.1

Yuichi Hirasawa, M.D.1

Kenichi Fukai, M.D.1

Tetsuhiro Chiba, M.D.1

Fumio Imazeki, M.D.1

Tatsuo Kanda, M.D.1

Mari Yatomi, M.D.3

Yuichi Takiguchi, M.D.3

Naohiko Seki, Ph.D.4

Hiromitsu Saisho, M.D.1

Takenori Ochiai, M.D.5

1 Department of Medicine and Clinical Oncology,Graduate School of Medicine, Chiba University,Chiba, Japan.

2 21st Center Century of Excellence Program,Graduate School of Medicine, Chiba University,Chiba, Japan.

3 Department of Respirology, Graduate School ofMedicine, Chiba University, Chiba, Japan.

4 Department of Functional Genomics, GraduateSchool of Medicine, Chiba University, Chiba, Ja-pan.

5 Department of Academic Surgery, GraduateSchool of Medicine, Chiba University, Chiba, Ja-pan.

The authors thank Professor Masaki Takiguchi andtheir colleagues for help and discussions.

Address for reprints: Osamu Yokosuka, M.D., De-partment of Medicine and Clinical Oncology (K1),Graduate School of Medicine, Chiba University,Inohana 1-8-1, Chiba-City 260-8670, Japan; Fax:(011) 81 432262088; E-mail: yokosukao@faculty.chiba-u.jp

Received September 7, 2005; revision receivedDecember 6, 2005; accepted January 12, 2006.

BACKGROUND. 5-Aza-2�-deoxycytidine (5-AzaC) is known well for its demethylation

effect and is a promising anticancer agent. However, to the authors’ knowledge,

serial changes in gene expression over time after 5-AzaC treatment have not been

studied to date. To clarify the categories of genes that are up-regulated or down-

regulated after 5-AzaC treatment, the authors surveyed the genes that had expres-

sion levels changed by 5-AzaC treatment in 6 hepatoma cell lines (Hep3B, HLE,

Huh7, HepG2, PLC/PRF/5, and Huh6).

METHODS. Cell lines were grown in medium that contained 1 �M of 5-AzaC.

Changes in messenger RNA levels were monitored from 24 hours up to 120 hours

after 5-AzaC treatment using an in-house microarray that consisted of 4608 com-

binational DNAs. Using clustering analysis to identify the genes that had gradually

changed expression levels and to exclude the substantial experimental noise by

microarray analysis, the authors focused on 206 up-regulated genes and 248

down-regulated genes.

RESULTS. According to their functional characterization, genes that were involved

in the cytoskeleton and the extracellular matrix were enriched significantly in the

up-regulated genes. Conversely, genes that were involved in metabolism were

enriched significantly in the down-regulated genes.

CONCLUSIONS. The current results demonstrated that 5-AzaC can regulate the

expression of groups of genes with characteristic functions. Cancer 2006;106:

2514 –25. © 2006 American Cancer Society.

KEYWORDS: gene expression, 5-aza-2�-deoxycytidine, microarray, cancer cell lines.

The inactivation of tumor suppressor genes occurs by mutation,loss of heterozygosity, and epigenetic silencing. Aberrant pro-

moter methylation is a fundamental mechanism of epigenetic silenc-ing.1– 4 Nearly 50% of all proven tumor suppressor genes can besilenced by hypermethylation along with many genes that play puta-tive roles in antitumor activities.5 5-Aza-2�-deoxycytidine (5-AzaC)has been used as an anticancer agent for patients with chronic my-elogenous leukemia in expectation of gene reactivation through itsdemethylation properties.6 In vitro administration of 5-AzaC cancause a rapid loss of DNA methyltransferase activity, and the hypom-ethylation of genes in vivo was observed after 5-AzaC administra-tion.6 –9 However, the effects that 5-AzaC has on malignant tumors areunclear and are of considerable interest. We speculate that manygenes are influenced by the administration of 5-AzaC through directepigenetic mechanisms that, in turn, affect subsequent genes indi-rectly; however, the global spectrum of genes influenced by 5-AzaCtreatment, both directly and indirectly, is not known.7,10 This sequen-

2514

© 2006 American Cancer SocietyDOI 10.1002/cncr.21905Published online 28 April 2006 in Wiley InterScience (www.interscience.wiley.com).

tial activation of genes and the resulting protein inter-actions may result in an anticancer effect. Therefore, itis very important to follow the expression changes ofmany genes to clarify the complex interactions thatcause the 5-AzaC effect. With this objective in mind,we selected microarray analysis, which can evaluatethe expression level of many genes at once, as anoptimal way to determine which genes are affected by5-AzaC. In previous studies, we evaluated the genes inhepatoma cells that were up-regulated after 5-AzaCadministration.11,12 To gain a more comprehensiveview of the sequential gene changes caused by 5-AzaCtreatment, we evaluated gene expression levels at sev-eral time points posttreatment by microarray analysis.In the current study, we monitored the changes inmessenger RNA (mRNA) levels every 24 hours up to120 hours after 5-AzaC treatment. From this analysis,we selected the genes that were up-regulated (n � 206genes) and down-regulated (n � 248 genes) with grad-ual changes, because we used the rationale that theeffect of hypomethylation by 5-AzaC would occurgradually. We focused on these candidate genes todetermine which categories of genes were affected by5-AzaC treatment.

MATERIALS AND METHODSCell Lines and RNA PreparationHepatoma cell lines (HLE, Huh7, HepG2, PLC/PRF/5,and Huh6) were purchased from Health Science Re-search Resources Bank, and Hep3B cells were ob-tained from the Cell Resource Center for BiomedicalResearch. Cells were maintained in Dulbecco Modi-fied Eagle Medium (DMEM) (Sigma-Aldrich Com-pany, St. Louis, MO) supplemented with 10% fetalbovine serum (Sanko Junyaku Company, Ltd., Tokyo,Japan). Just before use, 5-AzaC (Sigma-Aldrich Com-pany) was dissolved in DMEM. Cells were grown inmedium that contained 1 �M 5-AzaC for 1 to 5 days,and the medium and drug were replaced every 24hours. Control cells were cultured similarly but with-out 5-AzaC. Total RNA was prepared from the celllines after 24 hours, 48 hours, 72 hours, 96 hours, and120 hours by using Trizol reagent (Invitrogen Com-pany, Carlsbad, CA) according to the manufacturer’sinstructions. To clarify the effect of various concentra-tions of 5-AzaC in detail, 5-AzaC treatments at 0.5 �M,1.0 �M, 2.0 �M, and 5.0 �M for 96 hours were per-formed in the same manner, and total RNA was pre-pared from each sample.

Preparation of the cDNA MicroarrayA cDNA microarray chip, which consisted of 4608 cD-NAs, was made as described previously.13 Briefly, 4608unique clones were selected from approximately 9000

sequenced clones in an oligo-capped cDNA library ofhuman liver and stomach samples.14 Polymerase chainreaction (PCR)-amplified cDNA products were spottedonto carbodiimide-coated glass slides.

Microarray AnalysisFluorescent cDNA probes labeled with indocarbocya-nine (Cy3) or indodicarbocyanine (Cy5) were preparedfrom 20 �g of total RNA. Hybridization and fluores-cence detection were performed essentially as de-scribed previously.13 Images were analyzed withQuant Array (GSI Lumonics, Nepean, Canada) accord-ing to the manufacturer’s instructions. The mean andstandard deviation (SD) of background levels werecalculated for each time point, and the genes that hadintensities less than the mean � 2 SD of backgroundlevel at least at 1 time point were excluded from fur-ther analysis. The Cy5/Cy3 ratios for all spots on themicroarray were normalized by dividing them by themedian values. Genes with expression levels that var-ied more or less than the mean � 2 SD of all analyzedgenes at any of the time points were subjected tofurther analysis. For each cell line’s microarray data,genes that were expressed differentially after 5-AzaCtreatment were subjected to clustering analysis basedon expression patterns, and they were classified into 5or 7 clusters by using Avadis™ software (Hitachi Soft-ware Engineering Company, Yokohama, Japan).

Analysis of mRNA Expression by Quantitative ReverseTranscriptase-PCR in Cancer Cell LinesFirst-strand synthesis of cDNA was performed with theSuperscript First Strand Synthesis System for reversetranscriptase PCR (Invitrogen Company) according tothe manufacturer’s instructions. The cDNA templateswere synthesized from 1 �g total RNA. Quantitative PCRamplification was performed 3 times by using the ABIPRISM� 7000 Sequence Detection System (Applied Bio-systems, Foster City, CA) according to the manufactur-er’s instructions with the TaqMan� probe and primersets provided by Applied Biosystems. PCR amplificationconditions were as follows: preincubation at 50°C for 2minutes and 95°C for 10 minutes and 40 cycles at 95°Cfor 15 seconds and 60°C for 60 seconds. The expressionof glyceraldehyde-3-phosphate dehydrogenase was usedas a control.

Annotation of Gene FunctionTo identify the mechanism of 5-AzaC’s effect, genesthat were expressed differentially between treated andcontrol cell lines were classified by using 2 methodsbased on their function. The first method was ouroriginal classification into 16 groups according to theinformation of Entrez Gene (National Center for Bio-

Gene Expression Change by Demethylation/Arai et al. 2515

technology Information; available at URL: http://www.ncbi.nlm.nih.gov/[accessed April 2006]): Thosegroups were cell cycle, tumor suppressor, translationand processing, transcription and processing, signaltransduction, secreted proteins, oncogene, mitochon-drion, metabolism, immune response, enzyme, cy-toskeleton and cell membrane, cell growth and main-tenance, apoptosis, others, and expressed sequencetags (EST).13,15 The second method of classificationused the FatiGo Data-Mining program (available atURL: http://fatigo.bioinfo.cipf.es [accessed April2006]). This program automatically defined the molec-ular function of the genes based on the Gene Ontologycodes and showed the proportion (%) of genes in afunctional group among all groups.16 Using the FatiGodata-Mining program, we compared the frequency in4th-level terms of biologic process or molecular func-tion between genes that were up-regulated and down-regulated.

To clarify the statistical significance of the frequen-cies of the genes in each functional group, the Fisherexact test was performed using StatView (SAS InstituteInc., Cary, NC). P values �.05 were considered signifi-cant. We evaluated whether there were CpG islands inthe promoter region of the up-regulated genes (from� 500 to 0 base pairs) by using CpG Island Searcher(available at URL: http://ccnt.hsc.usc.edu/cpgislands2/[accessed April 2006]).

RESULTSMicroarray AnalysesAn in-house microarray that contained 4608 cDNAclones derived from human liver and stomach tissuewas used. The numbers of genes with meaningfulexpression at all time points were 1550, 2045, 1879,1982, 2272, and 1811 genes in the cell lines Hep3B,Huh6, PLC/PRF/5, HepG2, HLE, and Huh7, respec-tively. We designated genes with meaningful changes

FIGURE 1. Cluster analyses are

shown of genes that had changed ex-

pression levels in 6 hepatoma cell

lines after treatment with 5-aza-2�-

deoxycytidine (5-AzaC). Messenger

RNA (mRNA) levels were assessed at

24 hours (h), 48 hours, 72 hours, 96

hours, and 120 hours after 5-AzaC

treatment by microarray analysis.

Genes that were up-regulated or

down-regulated more than the

mean � 2 standard deviations at least

at 1 time point after 5-AzaC treatment

in the cell lines Hep3B (327 genes),

Huh6 (269 genes), PLC/PRF/5 (252

genes), HepG2 (257 genes), HLE (189

genes), and Huh7 (299 genes) were

subjected to hierarchical clustering

analysis. Time points are represented

in rows, and genes are listed in the

first column. Shading represents

higher (white), equal (gray), and lower

(black) mRNA levels relative to control

levels. White bars under each data set

indicate gene sets of gradually up-

regulated genes; black bars indicate

gene sets of gradually down-regulated

genes.

2516 CANCER June 1, 2006 / Volume 106 / Number 11

TABLE 1The Genes Gradually Up-Regulated or Down-Regulated in Greater than Two Hepatoma Cell Lines after 5-Aza-2�-Deoxycytidine Treatment*

GeneGeneID No. Symbol Function

Cell Lines

Hep3B Huh6 PLC/PRF/5 HepG2 HLE Huh7

Transgelin 2 8407 TAGLN2 Cytoskeketon � � � � � �

Adaptor-related protein complex2, � 1 subunit 1173 AP2M1 CG � � � �

Tropomyosin 1 (�) 7168 TPM1 Cytoskeleton � � � �

Thymosin, � 4, X chromosome 7114 TMSB4X Cytoskeketon � � � �

Thymosin, � 10 9168 TMSB10 Cytoskeketon � � � �

Myosin, light polypeptide 9,regulatory 10398 MYL9 Cytoskeketon � � � �

Actinin, � 4 81 ACTN4 Cytoskeleton � � � �

Peroxiredoxin 1 5052 PRDX1 CG � � �

Glutathione S-transferase � 2950 GSTP1 CG � � �

Connective tissue growth factor 1490 CTGF CG � � �

Tubulin, � 2 7280 TUBB2 Cytoskeketon � � �

Tubulin, � 3 7846 TUBA3 Cytoskeketon � � �

Syndecan 4 (amphiglycan,ryudocan) 6385 SDC4 Cytoskeketon � � �

Keratin 8 3856 KRT8 Cytoskeketon � � �

EST (BC001209) EST � � �

EST (AX014877) EST � � �

Pyruvate kinase, muscle 5315 PKM2 Metabolism � � �

Fascin homolog 1, actin-bundling protein 6624 FSCN1 Metabolism � � �

Mitochondrial carrier homolog 1(C. elegans) 23787 MTCH1 Mitochondrion � � �

CD151 antigen 977 CD151Signal

transduction � � �

Zyxin 22793 ZYXSignal

transduction � � �

Ras homolog gene family,member C 389 RHOC Transcription � � �

Proteasome (prosome,macropain) 26S subunit, non-ATPase, 2 5708 PSMD2 Translation � � �

Heat shock 90-kDa protein 1, � 3320 HSPCA Translation � � �

Heat shock 70-kDa protein 1A 3303 HSPA1A Translation � � �

Caveolin 1, caveolae protein, 22kD 857 CAV1

Tumorsuppressor � � �

A disintegrin-like andmetalloprotease (reprolysintype) with thrombospondintype 1 motif, 1 9510 ADAMTS1 CG ● ● ●

Dual specificity phosphatase 6 1848 DUSP6 CG ● ● ●

Source of immunodominantMHC-associated peptides 201595 SIMP EST ● ● ●

EST (AC005829) EST ● ● ●

EST (BG428075) EST ● ● ●

Mitogen-activated protein kinasekinase kinase kinase 3 8491 MAP4K3

Signaltransduction ● ● ●

Pericentriolar material 1 5108 PCM1 Transcription ● ● ●

Ribosomal protein L3 6122 RPL3 Translation ● ● ●

ID: identification; CG: cell growth and maintenance; EST: expressed sequence tags; MHC: major histocompatability complex.

Gene names, gene ID numbers, gene symbols, molecular function, and names of cell lines are listed. Genes that are up-regulated in a particular cell line are denoted by a white circle (�), and genes that are

down-regulated are denoted by a black circle (●).

Gene Expression Change by Demethylation/Arai et al. 2517

if their expression levels varied more or less than themean � 2 standard deviations of all the analyzedgenes at any of the time points (24 hours, 48 hours, 72hours, 96 hours, and 120 hours) after 5-AzaC treat-ment. The numbers of genes with meaningful changeswere 327, 269, 252, 257, 189, and 299 genes in the celllines Hep3B, Huh6, PLC/PRF/5, HepG2, HLE, andHuh7, respectively. The genes with meaningfulchanges were subjected to clustering analysis basedon expression patterns (Fig. 1).

Based on clustering analysis, we selected genesthat were up-regulated or down-regulated graduallyafter 5-AzaC treatment (Fig. 1). The numbers of genesthat gradually were up-regulated were 41, 46, 60, 55,51, and 68 genes in the cell lines Hep3B, Huh6, PLC/PRF/5, HepG2, HLE, and Huh7, respectively; and thenumbers of genes that gradually were down-regulatedwere 59, 47, 52, 64, 30, and 58 genes, respectively. Intotal, the numbers of genes with gradual up-regulationor down-regulation in at least 1 hepatoma cell line after5-AzaC treatment were 206 and 248 genes, respectively.

The genes that were up-regulated gradually after 5-AzaCtreatment in more than 2 cell lines (26 genes) and thegenes that were down-regulated gradually in more than2 cell lines (8 genes) are listed in Table 1.

Validation of Microarray Data with the Quantitative RT-PCR MethodTo determine the validity of results obtained by themicroarray analysis, the following 5 genes were selectedand subjected to quantitative RT-PCR: actinin � 4(ACTN4, Hs00245168 [Assay ID of TaqMan� probe]), fi-bronectin 1 (FN1, Hs00365052), vitronectin (VTN,Hs00169863), CD151 antigen (CD151, Hs00170407), andinterleukin 8 (IL8, Hs00174103). Messenger RNA levels ofthese 5 genes were nearly almost comparable betweenthe microarray analysis and the quantitative RT-PCRanalysis (Fig. 2), suggesting the competency of microar-ray analysis for the detection of changes in gene expres-sion after 5-AzaC treatment.

FIGURE 2. These charts compare

the results obtained with microarray

and quantitative reverse transcriptase-

polymerase chain reaction (RT-PCR)

analysis for changes in mRNA levels

after 5-aza-2�-deoxycytidine (5-AzaC)

treatment. Messenger RNA levels at

various time points after 5-AzaC treat-

ment in microarray analysis (open cir-

cles) and quantitative RT-PCR analysis

(solid circles) are shown in hours (h)

for the following mRNAs: actinin � 4

(ANXA1), fibronectin 1 (FN1), vitronec-

tin (VTN), CD151 antigen (CD151), and

interleukin 8 (IL8).

2518 CANCER June 1, 2006 / Volume 106 / Number 11

Functional Classification of Gradually Up-Regulated andDown-Regulated Genes in Hepatoma Cell Lines Treatedwith 5-AzaCFunctional characterizations of the genes that gradu-ally were up-regulated or down-regulated by 5-AzaCtreatment in each of the hepatoma cell lines areshown in Figure 3A. The distribution patterns of genefunction were similar between the 6 cell lines for bothup-regulated genes and down-regulated genes. How-ever, the majority of gradually up-regulated genes fell

into unique functional categories compared withgradually down-regulated genes. There were signifi-cantly more members of the category “cytoskeletonand membrane” (P�.05) in the gradually up-regulatedgenes in the cell lines Hep3B, Huh6, PLC/PRF/5,HepG2, and Huh7, which were evaluated by Fisherexact test for high and low frequencies. With statisticaldifferences, there were many members of the category“secreted proteins” (Hep3B and HepG2 cell lines) and“metabolism” (Hep3B and PLC/PRF/5 cell lines;

FIGURE 3. (A) These charts illus-

trated the distribution of functionally

categorized genes that were up-regu-

lated and down-regulated by 5-aza-

2�-deoxycytidine (5-AzaC) treatment

in each cell line. The functions of the

up-regulated and down-regulated

genes were classified into 15 groups.

Frequencies of the functionally classi-

fied genes are shown for each cell line

(except the “expressed sequence

tags” group). Statistical significance

was evaluated by Fisher exact tests

for high and low frequencies. An as-

terisk indicates P�.05. (B) Sequential

changes of gene ratios are illustrated

in selected functional categories after

5-AzaC treatment. The averages of

mRNA levels for genes in the catego-

ries “cytoskeleton and membrane”

(diamonds), “secreted proteins” (solid

circles), “metabolism” (triangles), and

for all of the analyzed genes in each

cell line (open circles and dashed

lines) at various time points after

5-AzaC treatment are shown. Error

bars indicate the standard deviation.

Double asterisks: P�.01; a single as-

terisk: P�.05, as determined by the

Student unpaired t test, compared

with the expression levels in all of the

genes analyzed in each cell line.

Gene Expression Change by Demethylation/Arai et al. 2519

P�.05) in the gradually down-regulated genes. Foreach cell line, averages of ratios of the genes in thecategories “cytoskeleton and membrane,” “secretedproteins,” and “metabolism” and all of the genes thatwere analyzed are shown in Figure 3B. Members of thecategory “cytoskeleton and membrane,” “secretedproteins,” and “metabolism” had significantly differ-ent patterns of expression compared with the expres-sion levels of all genes in each cell line when they wereanalyzed with the Student unpaired t test. The genesin the category “cytoskeleton and membrane” espe-cially showed a gradually increasing pattern in the celllines Hep3B, HLE, PLC/PRF/5, and Huh6. Next, weclassified genes that had gradual up-regulation (206genes) or gradual down-regulation (248 genes) in atleast 1 hepatoma cell line after 5-AzaC treatment.

Again, there was a significant fraction of graduallyup-regulated genes in the category “cytoskeleton andmembrane” (P�.05), and there was a significant frac-tion of down-regulated genes in the categories “se-creted proteins” and “metabolism” (P�.05) (Fig. 4).

To complete the analysis, we also classified the206 gradually up-regulated and 248 gradually down-regulated genes by using the FatiGo Data-Mining pro-gram, which is a different method of functional clas-sification that is based on Gene Ontology (GO)information at Level 4 of the biologic process. By usingthis method, 130 genes in the up-regulated categoryand 127 genes in the down-regulated category couldbe classified. The proportion of genes in the functionalgroups and the P values between up-regulated anddown-regulated genes are shown in Figure 5 (only the

FIGURE 3. (continued)

2520 CANCER June 1, 2006 / Volume 106 / Number 11

groups with P values �.1 are shown). The genes thatfell within the group with P values �.08 are listed inTable 2. Among them, the frequency of members ofthe “cell organization and biogenesis” (GO code0016043) category in the gradually up-regulated genes(P�.01) was significant. There were many members ofthe “cellular metabolism” (GO code 0044237), “pri-mary metabolism” (GO code 0044238), and “biosyn-thesis” (GO code 000958) categories in the graduallydown-regulated genes that showed statistical differ-

ences (P�.01). The members of the categories “loco-motion” (GO code 0040011), “cell motility” (GO code0006928), “cell adhesion” (GO code 0007155), “posi-tive regulation of cellular process” (GO code 0048522),and “response to stress” (GO code 0006950) tended tobe frequent in the gradually up-regulated genes, al-though the results were not statistically significant(P � .06, P � .06, P � .067, P � .085, and P � .089, re-spectively). The members of the “cell motility” cate-gory were identical to members of the “locomotion”category.

The Effect of Various Concentrations of 5-AzaC on GeneExpression LevelsAmong the genes that were related to “cytoskeleton andextracellular matrix,” “locomotion,” and “cell motility,”we selected the following 7 genes and examined theirexpression levels by quantitative RT-PCR: ACTN4, FN1,VTN, CD151, IL8, connective tissue growth factor (CTGF,Hs00170014 [Assay ID of TaqMan� probe]), and hepato-cyte growth factor activator inhibitor 2/placental bikunin(serine protease inhibitor 2 [SPINT2], Hs00173936). Theexpression levels of ACTN4, FN1, VTN, CD151, IL8, CTGFand SPINT2, were up-regulated by 5-AzaC treatment atall concentrations: even at concentrations as low as0.5 �M (Fig. 6).

DISCUSSIONAberrant DNA promoter region methylation is studiedto determine the mechanism of epigenetically si-lenced genes that are involved in cancer.1– 4 One ap-proach for restoring the expression of epigeneticallysilenced cancer genes for therapeutic purposes is touse hypomethylating agents, such as 5-AzaC, that re-turn promoters to their properly unmethylated state7

and that induce cell cycle arrest.17 In fact, 5-AzaCreportedly had clinical activity in chronic myeloge-nous leukemias that were refractory to imatinib in aPhase II study.6 Although hypomethylation by 5-AzaCtreatment was observed in vitro and in vivo,8,11 todate, the downstream events of hypomethylation havenot been elucidated. Microarray analysis is a powerfultool with which to evaluate the expression levels ofmany genes at once. Using this technique, we deter-mined that SPINT2, FN1, CTGF, collagen Type I �, andinsulin-like growth factor binding protein 2 were si-lenced by promoter hypermethylation in human hep-atoma cell lines related to carcinogenesis in combina-tion with 5-AzaC treatment at 1 �M for 96 hours.11,12

We intended to use a uniform concentration of5-AzaC with competent demethylation effect and withless cytotoxic effect in various cell lines. By examiningthe cyclin-dependent kinase inhibitor 2A, SPINT2, nic-otinamide adenine dinucleotide phosphate diaphorase

FIGURE 4. This chart illustrates the distribution of functionally categorized

genes that were up-regulated and down-regulated by 5-aza-2�-deoxycytidine

treatment in at least 1 hepatoma cell line. The functions of the up-regulated

and down-regulated genes were classified as shown in Figure 3. The numbers

of the functionally classified genes are shown (except the “expressed sequence

tags” group). Statistical significance was evaluated by Fisher exact test for high

and low frequencies. Asterisks indicate P�.05.

FIGURE 5. This chart illustrates the proportion of genes in the functional

groups based on Gene Ontology information at Level 4 of biologic processes.

Only the groups that had specific frequencies with P values �.1 between

up-regulated genes (open bars) and down-regulated genes (solid bars) are

shown. Asterisks indicate P�.05, as determined by the FatiGo Data-Mining

program.

Gene Expression Change by Demethylation/Arai et al. 2521

dehydrogenase, quinone 1, TMS1/ASC, and estrogenreceptor genes,11,18 –21 it appeared that 5-AzaC admin-istration at doses from 0.5 �M to 1.0 �M were capableof inducing a demethylation effect. We investigatedthe cell viability of hepatoma cell lines after 5-AzaCtreatment at concentrations of 0.5 �M, 1.0 �M, 2.0�M, and 5.0 �M for 96 hours by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenly)-2-(4-sulfonyl)-2H-tetrazolium assay. Although the cellviability did not change significantly between 0.5 �Mand 5 �M concentrations of 5-AzaC in Hep3B andHepG2 cells, the cell viability at 2 �M or 5 �M de-creased significantly compared with cell viability at 0.5�M or 1 �M in Huh6, PLC/PRF/5, HLE, and Huh7 cells(P�.05; Mann–Whitney U test; data not shown), whichshowed that 5-AzaC had less cytotoxic effect at 0.5 �Mor 1.0 �M than at 2.0 �M or 5.0 �M. Of 0.5 �M or 1.0�M with less cytotoxic effect, we used 1.0 �M as theconcentration of 5-AzaC in the current study becauseof the possible higher demethylation effect comparedwith what is achieved with 0.5 �M.

In most studies that evaluated the effect of 5-AzaCtreatment, the changes in gene expression were inves-tigated only at a single time point. In these up-regu-lated or down-regulated genes, many genes were in-duced transiently by the cytotoxicity of 5-AzaC and byexperimental noise of the microarray analysis. To ex-clude the influence of genes that were induced tran-siently, we analyzed sequential changes in gene ex-pression after 5-AzaC treatment. We then focused onthe genes with expression levels that changed gradu-ally, because the effect of hypomethylation by 5-AzaCwould occur gradually.

It is noteworthy that the distribution of gene func-tion showed similar patterns for both up-regulatedand down-regulated genes despite the use of differentcell lines. In 5 of 6 hepatoma cell lines, there weresignificantly more members of the “cytoskeleton andmembrane” category among the up-regulated genes(Fig. 3A). The average expression level of all genes inthe “cytoskeleton and membrane” category also grad-ually increased in most cell lines (Fig. 3B). In agree-ment, the FatiGo Data-Mining program categorized asignificant fraction of the gradually up-regulatedgenes into the “cell organization and biogenesis” cat-egory. Fourteen of 31 genes in the “cell organizationand biogenesis” category in shown in Figure 5 be-longed to the “cytoskeleton and membrane” categoryin shown in Figure 3. We compared the frequency in4th-level terms of “molecular function” (the term “bi-ologic processes” was used in Fig. 5) between genesthat were up-regulated and down-regulated by usingthe FatiGo Data-Mining program in the same manner.Among genes that had gradual up-regulation, mem-

bers of the “cytoskeletal protein binding” categorywere significantly frequent (data not shown). There-fore, although different methods of gene function clas-sification were used, it is obvious that the genes re-lated to cell structure and to the extracellular matrixwere up-regulated by 5-AzaC treatment.

The down-regulation of genes related to the cy-toskeleton and the extracellular matrix may collapse thestructure of normal tissue or may change the phenotypeof cells. In addition, the disturbance of epithelial junc-tion caused by deficiencies in the extracellular matrixmay allow cancer cells to invade the surrounding tis-sue.22 In fact, the down-regulation of genes related to thecytoskeleton and the extracellular matrix was relatedstrongly to the activity of metastasis and invasion ofcancer cells,23–25 and the actin cytoskeleton and micro-tubules became new targets for anticancer therapies.26,27

It is noteworthy that genes in the “locomotion,” “cellmotility,” and “cell adhesion” categories, all of which areimportant activities for invasion and metastasis of ma-lignant cells, were observed frequently among the grad-ually up-regulated genes (P � .06). The expression levelsof 7 genes related to “cytoskeleton and extracellular ma-trix,” “locomotion,” and “cell motility” were up-regu-lated after 5-AzaC treatment at various concentrations(Fig. 6), indicating that the up-regulation of these geneswas not limited in 5-AzaC treatment at a concentrationof 1 �M. We speculate that the administration of 5-AzaCmay restore expression levels of the genes related to“cytoskeleton and extracellular matrix” to recover nor-mal structure and cell-to-cell communication. Conse-quently, this may inhibit the growth and invasion ofmalignant tumors and may restore normal cell growth.

The genes in the category “response to stress”,including IL8 and IL27, also were frequent among thegradually up-regulated genes (P � .089). In a reportthat studied the effect of 5-AzaC on pancreatic cancercell lines, there were many genes related to “defenseresponse” that were up-regulated by 5-AzaC adminis-tration. Furthermore, 5-AzaC induced the activity ofthe interferon signaling pathway.28 Together with ourresults, we infer that 5-AzaC may induce up-regulationof the genes related to stress response, for example,cytokines like interferon and interleukin.

Among the genes with gradual down-regulation,members of the “secreted proteins” and “metabolism”categories were observed frequently. The category “se-creted proteins” largely contains plasma proteins thatare biosynthesized in the liver, and the category “metab-olism” is composed mainly of enzymes involved in he-patic intermediary metabolism of compounds, such assugars, lipids, amino acids, nucleotides, steroids, andxenobiotics.13 Using the alternate FatiGo Data-Miningprogram, we reached the same result. Many of the grad-

2522 CANCER June 1, 2006 / Volume 106 / Number 11

TABLE 2Distribution of the Up-Regulated and Down-Regulated Genes Based on the Information from Gene Ontology

Up-Regulated Genes Down-Regulated Genes

Cell organization and biogenesis (GO code 16043)ACTN4 TESK2 KLF6 CXCL1 KIAA1797 FGFR2IGFBP3 CFL1 TUBB6 TEBP C18orf22 PDCD11IGFBP4 TMSB10 PLSCR1 FGFR1 SNX17 CAPZA2DDX5 RPLP0 TMSB4X POP4 HPS1HMGA1 TUBB2 IGFBP7GJA1 H3F3B KRT8FLNA SQSTM1 CTGFTUBB4 NAP1L4 PALM2-AKAP2PFN1 SMARCA1 FSCN1FLNB TUBA3 LCN7Cellular metabolism (GO code 44237)TESK2 IDS JUNB DHRS3 DUSP6 POLR3EKCNMA1 ENO1 TUBB6 CRSP7 EIF3S6IP RPL22RPS2 HSPB1 PKM2 CANX PSMA3 A2MND4L GPX2 RNF40 SCD ALDH4A1 IGF2IGFBP4 RPLP1 ERH ATF4 LIPF CYP2C8SFRS2 HSPCB PGAM1 PDK4 PPM1A C18orf22GPX1 RPLP0 HMGA1 ISGF3G TEBP AP3D1PRPF4 ITGB4BP TUBB2 APOA1 SEC63 ATP6V0CSERPINH1 TXNRD1 H3F3B DNMT1 MMP1 HMGCS2TTR GJA1 PRSS23 ADAMTS1 APOA2 TCF4MAP2K3 SFPQ TGM2 PDCD11 HSPA9B SHMT2VCP LDHA NDUFV2 GLUL OSBP2 GPISERPINE2 WBSCR1 IL27 STAT6 PLRG1 TSTSQSTM1 DNAJA1 CTGF ZNF614 MAP4K3 RPP14HSPCA RPSA TRIM28 YARS POMT2 FGFR1SCYE1 GPX3 NAP1L4 ATP5E DHPS NDUFS3POLD4 TUBB4 TUBA3 CYP2C9 MARS PCM1SMARCA1 HDLBP KLF6 AKR1C1 RPL3 ETV1LCN7 MSH6 HSPA1A SNX17 CAPZA2 POP4PGK1 PSMB3 BRSK2 CCNT2 USP34

TM4SF4 TPRT ME3BTD NSF PPGBSIMP NR4A1 ITIH2CLPP APOB USP15HKR3 UGT8 FGFR2HNRPA2B1 EIF4B SNRPD2BZW1 ASS MAT1A

Primary metabolism (GO code 44238)TESK2 IDS JUNB DHRS3 DUSP6 POLR3EKCNMA1 ENO1 TUBB6 CRSP7 EIF3S6IP RPL22RPS2 HSPB1 RPLP1 CANX PSMA3 ALDH4A1PKM2 IGFBP4 HSPCB SCD UGT2B10 LIPFRNF40 SFRS2 PGAM1 A2M ATF4 PPM1AERH RPLP0 HMGA1 IGF2 PDK4 C18orf22PRPF4 ITGB4BP TTR ISGF3G TEBP DNMT1SERPINH1 TUBB2 MAP2K3 APOA1 SEC63 ADAMTS1GJA1 H3F3B VCP MMP1 ATP6V0C PDCD11SFPQ PRSS23 SERPINE2 APOA2 HMGCS2 GLULLDHA TGM2 NEU1 HSPA9B TCF4 STAT6WBSCR1 SQSTM1 HSPCA OSBP2 SHMT2 ZNF614DNAJA1 IL27 SCYE1 PLRG1 GPI POMT2RPSA CTGF TUBB4 MAP4K3 YARS DHPSTRIM28 POLD4 HDLBP RPP14 ATP5E NDUFS3NAP1L4 SMARCA1 LCN7 FGFR1 MARS SNX17PSMC3 TUBA3 PGK1 RPL3 PCM1 BRSK2MSH6 KLF6 HSPA1A CAPZA2 ETV1 TM4SF4PSMB3 CCNT2 POP4 NSF

(continued)

Gene Expression Change by Demethylation/Arai et al. 2523

ually down-regulated genes were in the “cellular metab-olism,” “primary metabolism,” and “biosynthesis” cate-gories (Fig. 5). The genes in these categories were relatedstrongly to metabolism and biosynthesis, which are es-sential for the basic activities of hepatocytes. The down-regulation of these genes may indicate that 5-AzaC mostlikely has the ability to inhibit the basic activity of cells.This may explain why 5-AzaC has side effects when it isused as a therapeutic drug.

Although we focused on changes in gene expres-sion in the current study, finding a new tumor sup-pressor gene would help to elucidate the mechanismof hepatocellular carcinogenesis and would identify anew target for cancer therapy. In hepatocellular car-cinoma (HCC), the inactivation of cyclin-dependentkinase inhibitor 2A and glutathione S-transferase P1 byepigenetic factors has been reported.29,30 We previ-ously reported that the SPINT2 gene also was silencedby an epigenetic mechanism in HCC and could sup-press cell growth in hepatoma cell lines using a mi-croarray with relatively small numbers of genes.11 Inthat study, SPINT2, phospholipid scramblase 1(PLSCL1), and caveolin 1 (CAV1) were included in thecategory “tumor suppressor,” and we found that there

FIGURE 6. This graph illustrates gene expression levels after various con-

centrations of 5-aza-2�-deoxycytidine (5-AzaC). Messenger RNA levels of 7

genes were measured 96 hours after 5-AzaC treatment at various concentra-

tions (0.5-5.0 �M) by using quantitative reverse transcriptase-polymerase

chain reaction analysis. Asterisks indicate that the ratios of expression levels

were shown in logarithm (base 2). Error bars indicate the standard deviation.

ACTN4: actinin � 4; FN1: fibronectin 1; VTN: vitronectin; CD151: CD151

antigen; IL8: interleukin 8; CTGF: connective tissue growth factor; SPINT2:

serine protease inhibitor 2.

TABLE 2(continued)

TPRT USP34 NR4A1

ME3 SIMP APOBPPGB CLPP UGT8ITIH2 HKR3 EIF4BUSP15 HNRPA2B1 ASSFGFR2 BZW1 MAT1ASNRPD2

Biosynthesis (GO code 9058)RPS2 HSPB1 RPLP0 EIF3S6IP RPL22 GPT2RPLP1 HSPCB IL27 SCD ALDH4A1 ATP6V0CITGB4BP WBSCR1 SCYE1 TEBP AP3D1 GLULRPSA APOA2 HMGCS2 POMT2

GPI YARS MARSATP5E DHPS TPRTRPL3 TM4SF4 EIF4BSIMP UGT8 ASS

BZW1Locomotion (GO code 40011) and cell motility (GO code 6928)*ACTN4 IL8 ATP1A1 YARS CAPZA2SPINT2 SERPINE2 FLNAARPC1B CTGF FN1Cell adhesion (GO code 7155)TESK2 FLRT3 FLRT1 DSC2 COL6A2 THBS2CD151 OMD ZYX NID CSPG2 PPFIA1LGALS3BP ARHGDIA IL8TGM2 RPSA CTGFGPR56 FN1 VTN

GO: Gene Ontology.

* Function, GO codes, and gene symbols are listed. The genes listed in the category “Cell motility” were identical to those for “Locomotion”.

2524 CANCER June 1, 2006 / Volume 106 / Number 11

were CpG islands in the promoter regions of these 3genes. Furthermore, CAV1 was down-regulated insmall cell lung cancer, and its promoter region washighly methylated.31 Reactivation of the expression ofPLSCL1 or CAV1 by demethylation may inhibit theproliferation of HCC directly.

Monitoring the changes in mRNA levels up to 120hours after 5-AzaC treatment allowed us to select forgenes that had gradual changes in expression levels. Wealso were able to classify these genes into distinct cate-gories of function. 5-AzaC may induce the expression ofgenes that are important for the cytoskeleton and theextracellular matrix. In contrast, it may inhibit genesrelated to metabolism and biosynthesis. This search forgenes that gradually change after 5-AzaC treatment mayprovide clues about the anticancer effects of 5-AzaC.

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