ARTICLES

Identification of Distinct and Common GeneExpression Changes After Oxidative Stressand Gamma and Ultraviolet Radiation

Alexandra N. Heinloth,1 Rodney E. Shackelford,1 Cynthia L. Innes,1 Lee Bennett,2 Leping Li,3

Rupesh P. Amin,2 Stella O. Sieber,2 Kristina G. Flores,1 Pierre R. Bushel,2 and Richard S. Paules1,2*1Growth Control and Cancer Group, National Institute of Environmental Health Sciences, Research Triangle Park,North Carolina2NIEHS Microarray Center, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina3Biostatistics Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina

The human genome is exposed to many different kinds of DNA-damaging agents. While most damage is detectedand repaired through complex damage recognition and repair machineries, some damage has the potential to escapethese mechanisms. Unrepaired DNA damage can give rise to alterations and mutations in the genome in an individualcell, which can result in malignant transformation, especially when critical genes are deregulated. In this study, we

investigated gene expression changes in response to oxidative stress, gamma (g) radiation, and ultraviolet (UV)radiation and their potential implications in cancer development. Doses were selected for each of the threetreatments, based on their ability to cause a similar G1 checkpoint induction and slow down in early S-phase

progression, as reflected by a comparable reduction in cyclin E–associated kinase activity of at least 75% inlogarithmically growing human dermal diploid fibroblasts. To investigate gene expression changes, logarithmicallygrowing dermal diploid fibroblasts were exposed to either g radiation (5 Gy), oxidative stress (75 mM of tert-butyl

hydroperoxide (t-butyl-OOH)), or UV radiation (UVC) (7.5 J/m2) and RNA was harvested 6 h after treatment. Geneexpression was analyzed using the NIEHS Human ToxChip 2.0 with approximately 1901 cDNA clones representingknown genes and expressed sequence tags (ESTs). We were able to identify common and distinct responses in dermal

diploid fibroblasts to the three different stimuli used. Within our analysis, gene expression profiles in response to gradiation and oxidative stress appeared to be more similar than profiles expressed after UV radiation. Interestingly,equivalent cyclin E–associated kinase activity reduction with all the three treatments was associated with greatertranscriptional changes after UV radiation than after g radiation and oxidative stress. While samples treated with UV

radiation displayed modulations of their mitogen activated protein kinase (MAPK) pathway, g radiation had its majorinfluence on cell-cycle progression in S-phase and mitosis. In addition, cell cultures from different individuals displayedsignificant differences in their gene expression responses to DNA damage. Published 2003 Wiley-Liss, Inc.{

Key words: gene expression; DNA damage; oxidative stress

INTRODUCTION

By the year 2050, the total number of cancer caseswithin theU.S. population is expected todouble. Themain reason for this development is the demo-graphic phenomenon of an increasing aging popula-tion [1]. In addition to hereditary predispositions, amajor risk factor for the development of cancer isexposure toenvironmentalcarcinogens.Basic tocan-cer development is damage of DNA and failure torepair the damage or to undergo apoptosis. Twosources for DNA damage are ultraviolet (UV) radia-tion [2] and gamma (g) radiation [3,4]. Exposure to gradiation results in specific damage to the DNA, e.g.,formation of uracil, abasic sites, 8-oxoguanine,single-strand breaks, and double-strand breaks [5].Repair of double-strand breaks involves recombina-tional processes (homologous recombination and

MOLECULAR CARCINOGENESIS 37:65–82 (2003)

Published 2003 WILEY-LISS, INC. yThis article is a US Government workand, as such, is in the public domain in the United States of America.

Rodney E. Shackelford’s present address is Des Moines Universityof Osteopathic Medicine, Des Moines, IA 50309.

*Correspondence to: Growth Control and Cancer Group,National Institute of Environmental Health Sciences, Mail Drop D2-03, P. O. Box 12233, 111 Alexander Drive, Research Triangle Park,NC 27709.

Received 14 January 2003; Revised 13 January 2003; Accepted 17January 2003

Abbreviations: UV, ultraviolet; BER, base excision repair; ROS,reactive oxygen species; t-butyl-OOH, tert-butyl hydroperoxide; EST,expressed sequence tag; MAPK, mitogen activated protein kinase;DTT, dithiothreitol; SDS, sodium dodecyl sulfate; SSC, saline sodiumcitrate; CV, coefficient of variation; RT, reverse transcription; PCR,polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphatedehydrogenase; CDC20, cell division cycle 20; GA/KNN, geneticalgorithm/K nearest neighbor; PCA, principal component analysis;PC#1, principal component 1; PC#2, principal component 2; CDC,cell division cycle; MCM, minichromosome maintenance; CDK,cyclin dependent kinase; RFC, replication factor C; PCNA, prolifer-ating cell nuclear antigen; FEN1, flap structure-specific endonuclease1; MAD2, mitotic arrest deficient 2; NEK2, NIMA-related kinase 2.

DOI 10.1002/mc.10122

nonhomologous end-joining) [6]. Base excisionrepair (BER) is activated by base modifications andsingle-strand breaks [7]. Gamma radiation inducesthe generation of reactive oxygen species (ROS) [8];one subcomponent of DNA damage induced by gradiation is ascribed to ROS. Besides the exogenousgeneration of oxidative stress from g radiation, endo-genous ROS are constantly produced by mitochon-drial oxidative phosphorylation and themetabolismof exogenous compounds [9]. In contrast, UV radiat-ion (UVB and UVC) induces 6-4 photoproducts,bulky adducts, and cyclobutane pyrimidine dimers[5]. These DNA lesions activate nucleotide–excisionrepair pathways [10,11].It was our goal to compare and contrast the

cellular responses to different kinds of DNA-dama-ging agents. We used UVC radiation instead of UVB(even though UVC is absorbed by the ozone layer) asan efficient DNA-damaging agent in order to pro-duce bulky DNA lesions to compare and contrastwith cellular responses to DNA damage generatedfollowing exposure to g radiation and tert-butylhydroperoxide (t-butyl-OOH). In order to profiletranscriptional responses to DNA-damaging agentsas directly as possible, we investigated gene expres-sion changes 6 h after exposure, after a pilot studyshowed a lack of a significant effect 2h after exposureto g radiation (data not shown).In the present study, we utilized cDNAmicroarray

analyses to investigate gene expression changes afterg irradiation, oxidative stress, and UV irradiation inhuman dermal diploid fibroblasts to elucidate dis-tinct and common cellular responses induced byexposure to these different forms of DNA-damagingagents. t-Butyl-OOH was used as a source for ROSbecauseof its resistance tohydrolysis by catalase [12].We avoided the use of a peroxide that could behydrolyzed by catalase and in turn could introduceexperimental variation based on the differencesof catalase activities in cell cultures from differentindividuals.Analysis of gene expression changes is a step

towards the identification of potential biomarkersof exposure to these different DNA-damaging agentsand further clarification about which cellular re-sponses are activated by the selected treatments. Weutilized a custom-built cDNA microarray chip with1901 cDNA clones of known genes and expressedsequence tags (ESTs), including a selection of genesimplicated in cell-cycle regulation, DNA replication/repair, and signal transduction pathways, includingmitogen activated protein kinase (MAPK) pathwayregulation [13]. In order to apply doses of theseDNA-damaging agents that resulted in comparable biolo-gical consequences, we normalized each treatmentto inhibition of cyclin E-associated kinase activity,as a parameter for G1 checkpoint induction and pro-gress through early S-phase. We chose this normal-ization approach because all the three different

treatments induce a similar early G1 checkpoint.This is in contrast to the G2 checkpoint responseto these agents in which UV irradiation fails to prod-uce the strong, rapid G2 delay observed followingexposure to g radiation. In this study we were inter-ested in rapid signaling events involved in cell-cyclecheckpoint responses to DNA damage. We furtherinvestigated the effect of inter-individual differencesby utilizing three different human dermal diploidfibroblast cell cultures in our experimental design.We found that all three different treatments

activated very distinct gene expression patterns,e.g., g radiation had major impact on expression ofgenes involved in DNA replication and mitosis,while UV radiation changed the expression of genesinvolved in MAPK pathways. Additionally, thedifferent genetic background of single individualshad major effects on their gene-expression responseto different DNA-damaging agents.

EXPERIMENTAL PROCEDURES

Cell Cultures and Culture Conditions

GM03349, GM08400, and GM08402, humandermal diploid fibroblast cells, were obtained fromthe National Institute of General Medical SciencesHuman Genetic Mutant Cell Repository (Camden,NJ). The donor of GM03349 was a 10-yr-old male; ofGM08400, a 37-yr-old female; and of GM08402, a32-yr-old male. These cells were used at passages15–19. Cells were grown at 378C in a humidified 5%CO2 atmosphere in Dulbecco’s modified Eagle’smedium, supplemented with 10% fetal bovineserum (Life Technologies, Gaithersburg, MD) and2 mM of L-glutamine (Life Technologies).Logarithmically growing cell populations in 100

mm plastic dishes (Becton Dickinson Labware,Franklin Lakes, NJ) were treated as follows. For gradiation, treatment cells were exposed to g-rays atroom temperature using a 137Cs source at a rate of2.6Gy/min for a final dose of 5Gy. Cells treatedwithoxidative stress were exposed to t-butyl-OOH for15 min. The cultures were then washed 2� withwarmmedium and the preconditionedmediumwasreturned to the cultures. In experiments in whichsome cell cultures were treatedwith t-butyl-OOH, allplates within the same experiment were washed asdescribed above. For UV-irradiated cells, the culturemedium was removed and the cells were exposed to7.5 J/m2 at 254 nm (UVC), then the preconditionedmedium was returned. UV-mock treated cells werehandled the same way, except the UV lamp was leftoff. After treatment, the cells were incubated for thetimes indicated and harvested.

P33CDK2/Cyclin E Histone H1 In Vitro Kinase Assay

At the indicated times after treatment, cells wereharvested and solubilized on ice in kinase lysis buffer(10 mM sodium phosphate (pH 7.2), 150 mM NaCl,

66 HEINLOTH ET AL.

1% NP-40, 1 mM EDTA, 5 mM EGTA, 5 mM b-glycerophosphate, 1 mM dithiothreitol (DTT), and10 mg/mL leupeptin). Protein concentration wasdetermined using a detergent-compatible proteinassay kit (Bio-Rad Laboratories, Inc., Melville, NY)with bovine serum albumin as a standard. Fivehundred micrograms of protein was used per kinasereaction in a total volume of 500 mL. Immunopreci-pitations were performed with 4 mL of antihumancyclin E (Cat. #05-363 Upstate Biotechnology, Inc.,Lake Placid, NY). After preclearing with protein-Gagarose beads (Life Technologies), samples wereincubated with primary antibody for 2 h and thenprecipitated with protein-G agarose beads. Kinasereactionswere performed inhistoneH1kinase buffer(20mMHEPES (pH7.3), 80mM b-glycerophosphate,20 mM EGTA, 50 mM MgCl2, 5 mM MnCl2, 1 mMDTT, 10 mg/mL leupeptin, 10 mM cyclic AMP-dependent protein kinase-inhibitory peptide) with5 mg of histone H1 and 5 mCi [32g-P]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech, Piscataway,NJ) for 30 min at 348C. Reactions were stopped byaddition of 2� sodium dodecyl sulfate (SDS) samplebuffer (4% SDS (lauryl sulfate), 150 mM of Tris (pH6.8), 20% glycerol, 100 mM of DTT, 0.02% bromo-phenol blue) and boiled for 5 min; proteins wereresolved by 12% SDS–polyacrylamide gel electro-phoresis.After stainingwithCoomassieblue toverifyequal histone protein loading, gels were dried andautoradiography was performed with Hyperfilm MP(Amersham Pharmacia Biotech). For quantification,gels were analyzed with Molecular Dynamics Phos-phorImager and Image-Quant software. All kinaseassays were performed at least in triplicate.

RNA Preparation

Six hours after treatment, cells were harvested,pelleted, and flash frozen. RNA was isolated withQiagen RNeasy Midi Kit (Qiagen, Inc., Valencia, CA)according to the instructions of the manufacturer.

cDNA Microarray

For labeling of target to analyze mRNA expressionlevels, cDNA was generated by in vitro transcriptionfrom 35 mg to 75 mg total RNA. RNA was combinedwith 1 mg of oligo dT12–18 primer (AmershamPharmacia Biotech) and 10 U of RNAse-inhibitor(Invitrogen, Carlsbad, CA) before heating for 10minat 708C. After chilling on ice for 2 min, first strandbuffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mMMgCl2) (Invitrogen), 11 mM DTT (Invitrogen),2.2 nM FluoroLink Cy3-deoxy (d) UTP or Cy5-dUTP(Amersham Pharmacia Biotech), dNTP mix (0.7 mMdATP, 0.7 mM dGTP, 0.7 mM dCTP, 0.4 mM dTTP)(Amersham Pharmacia Biotech), and 2 mL of SUPER-SCRIPTTM II Reverse Transcriptase (Invitrogen) wereadded. Samples were incubated at 428C for 1.5 hbefore addition of another 2 mL of SUPERSCRIPTTM IIReverse Transcriptase and incubation for another

1.5h.Todegrade theRNAafter cDNAsynthesis, 30mLof of 0.1 M NaOH was added and incubated for30 min at 708C. Addition of 30 mL of 0.1 M HClneutralized the pH, and Cy3-dUTP and Cy5-dUTPlabeled samples were pooled. Unincorporated labelwas removed by using Microcon-30 filters (MilliporeCorp., Bedford, MA). To limit nonspecific binding ofthe probe, 10 mg of human COT1 DNA (Invitrogen)per 10mgof RNAand20 mgof yeast tRNA (Invitrogen)were added. Probes were added to a hybridizationsolution (3� saline sodium citrate (SSC), 2� Den-hardt’s, and 0.8% SDS) and, after boiling for 2 min,purified through a 0.45 mm filter (Millipore). Thepretreated labeled target cDNA representative of thecellular mRNA was applied to a glass cDNA micro-array chip, covered by a coverslip, and incubated for24 h in a humidified chamber at 658C. The utilizedcustom-built chips (ToxChip version 2.0) contained1901 known human genes and ESTs [14]. The cDNAclones on this chip were sequence verified andannotated according to UniGene [15].To remove the coverslip, slides were inverted in

0.5� SSC, 0.01% SDS for 5 min, washed in 0.5� SSC,0.01% SDS for 5 min, and in 0.06� SSC for 5 min.Thereafter slides were dried by spinning for 3 min at1000 rpm. Each RNA sample was labeled and hybri-dized on four independent replicate microarraychips, including fluor reversals (twice mock RNAwas labeled with Cy3-dUTP and treated RNA withCy5-dUTP, twice treated RNA was labeled with Cy3-dUTP and mock RNA with Cy5-dUTP). Slides werescanned at 532 nm (Cy3-dUTP) and 635 nm (Cy5-dUTP) wavelengths with a GenePix 4000A scanner(Axon Instruments, Union City, CA) to generate16-bit gray scale pixel image files of the arrays.

Data Analysis

For acquisition and normalization of the signalintensities from the image files, we utilized IPLabsimage processing software (Scanalytics, Inc., Fairfax,VA) with the ArraySuite 2.0 extensions (NationalHuman Genome Research Institute) based on dataacquisition and analysis algorithm developed byYidong Chen [16,17]. These programs extract pixelintensity values from each scanned channel, com-pute ratio values from the signals, normalize theratios to the average ratios of all clones on the chip,and identify, based on a user defined confidencelevel, genes that demonstrate statistically significantexpression changes. Spots with an intensity valuebelow background level on either channel were ex-cluded from further analysis (in average 3% of allspots per chip). Genes identified at a 90% confidencelevel as being upregulated or downregulated werestored in MAPS, a database developed for manage-ment and statistical validation of replicate geneexpression data [18]. We used a binomial distribu-tion to detect occurrence of differentially expressedgenes among four independent hybridization trials,

GENE EXPRESSION PROFILES AFTER DNA DAMAGE 67

which included two analyses with fluor reversal, foreach RNA sample analysis. This resulted in a total of12 hybridizations per treatment from the threedifferent cell cultures. This analysis approach incre-ases the statistical confidence of the set of differen-tially expressed genes by minimizing the genesdetected as false positive. To insure that reportedgene expression changes were reproducible in morethan one tested individual, only gene expressionchanges at the 90% confidence level in at least five of12 hybridizations were considered for further analy-sis (the probability of detecting a differentially ex-pressed gene by chance is P¼0.003). To excludegenes that demonstrated a bias for a particularfluorophore or very variable expression, the coeffi-cient of variation (CV) for each gene across replicatehybridizations was calculated. The CV (standarddeviation/average absolute values of the calibratedratio) was determined using the log2 ratio intensityvalues of the genes detected as differentially expres-sedat agivenconfidence level.GeneswithaCVvaluegreater than 0.5 were eliminated from further stati-stical analysis.As a second approach to identify differentially

expressed genes, we utilized the mixed linear modelanalysis developed by Wolfinger et al. [19]. Thismethod analyzes the complete gene-set representedonour chips basedon reproducibility of theobservedgene expression changes across replicate measures.We considered genes being differentially expressedwhen the reproducibility of their change had a P-value of 0.005 or less, and an expression level thatwas either less than 0.8 or more than 1.2 of control.

Real-Time Reverse Transcription (RT)–PolymeraseChain Reaction (PCR)

Fivemicrograms of total RNA from eachmock andtreated sample was individually combined with 1 mgof oligo dT12–18 primer (Amersham PharmaciaBiotech) and 10 U of RNAse-inhibitor (Invitrogen)before heating for 5min at 658C. After chilling on icefor 2 min, we added first strand buffer (50 mM Tris-HCl, pH 8.3, 75mMKCl, 3mMMgCl2) (Invitrogen),11 mM DTT (Invitrogen), dNTP mix (0.7 mM dATP,0.7 mM dGTP, 0.7 mM dCTP, 0.7 mM dTTP)(Amersham Pharmacia Biotech), and 1 mL of SUPER-SCRIPTTM IIReverseTranscriptase (Invitrogen) to thesamples. During incubation at 428C for 1.5 h, cDNAwas produced. Samples were denatured by incuba-tion at 708C for 15 min. One twentieth of the result-ing cDNA was amplified in triplicate with genespecific primers (listed below) utilizing the SYBRGreen PCR Core Reagents (PE Applied Biosystems,Foster City, CA) according to the recommendationsof the manufacturer. The amplification reactionwas performed in a ABI Prism 7700 Sequence Detec-tor (PE Applied Biosystems) with the followingcycling conditions: initial incubation at 958C for10 min, 40 cycles of 958C for 15 sec, and 608C for

1 min. The threshold cycle value for each amplifica-tion product was determined with Sequence Detec-tion System software (PE Applied Biosystems). Thelevels of expression for each gene were normalizedusing glyceraldehyde 3-phosphate dehydrogenase(GAPDH) levels according to the comparative thres-hold cycle method. To ensure primer specificity, weperformed dissociation-curve measurements of theamplification product. Cycling conditions for dis-sociation-curves: 958C for 15 sec, 608C for 20 sec, and958Cfor 15 secwitha ramp timeof 19minand59 sec.GAPDH forward primer: CCA CCC ATG GCA AATTCC; GAPDH reverse primer: TGG GAT TTC CATTGATGACAAG;p21 forwardprimer:CCTAATCCGCCCACAGGAA; p21 reverse primer: AAGATGTAGAGC GGG CCT TTG; RFC4 forward primer: CTGCCA GTG CGG GAA GTA G; RFC4 reverse primer:ATC CAC ACA TTT TGG GCG ATA T; Heat shockprotein A5 forward primer: CCG CTT TTT AAG ATGGCC AAT; Heat shock protein A5 reverse primer:CCT AAG CGG CTG TTT ACT GCT T; CDC20forward primer: TGG GCT CAT GGT CAG ACT CA;CDC20 reverse primer: CCC CAT TAC AAG GAGCTC ATC T.

RESULTS

Reduction of Cyclin E–Associated Kinase Activity inHuman Dermal Diploid Fibroblasts by Oxidative Stress,g and UV Radiation

Inhibition of cyclin E–associated kinase activity isan integral component of bothG1 checkpoint induc-tion and inhibition of progression through S-phase[20,21]. To examine the relative biological conse-quences of oxidative stress and g and UV radiation,we compared their capability to induce a reductionof cyclin E–associated kinase activity in humandermal diploid fibroblasts. Our goal was to evaluategene expression changes associated with a compar-able, efficient reduction of cyclin E–associatedkinase activity, a reduction that we defined as beingefficient when the activity in treated samples wasreducedbyat least 75%as comparedwith the activityof control samples.Weapplieddifferent doses of either t-butyl-OOH, g

radiation, or UV radiation to normal human fibro-blasts and measured cyclin E-associated kinase acti-vity after 3 and 6 h. As shown in Figure 1, 75 mM oft-butyl-OOH, 5 Gy g radiation and 7.5 J/m2 UVradiation resulted in at least a 75% reduction ofcyclin E–associated kinase activity after 6 h. Thesedoses were used in the other experiments in thisstudy.Interestingly, reduction of cyclin E–associated

kinase activity after oxidative stress showed adifferent kinetic from that noted after g and UVradiation. While after 3 h, both g and UV radiationhad already induced a significant reduction, cellstreated with t-butyl-OOH were not yet inhibited in

68 HEINLOTH ET AL.

their cyclin E–associated kinase activity but reacheda similar level of reduction as seenwith UV radiation6 h after treatment.

With Equivalent Reduction of Cyclin E–AssociatedKinase Activity, More Transcriptional Changes After UV

Radiation Than After Oxidative Stress and g Radiation

Samples treated with oxidative stress, UV radia-tion, and g radiation were analyzed by cDNA micro-array technology in comparison to their time-matched mock-treated control samples. Statisticallysignificant (P� 0.005) gene expression changes wereidentified based on a mixed linear model analysis(Figure 1A of Web-based supplementary material tothis article, available at www.interscience.wiley.com), as well as a confidence level based analysisutilizing replicates and an estimation of false posi-tives based on a binomial distribution prediction of agene being identified as differentially expressed inreplicate analyses by totally random chance. Moregene expression changes were observed by bothstatistical approaches after UV radiation than after gradiation and oxidative stress. Comparison of totalnumbers of gene expression changes showed stron-ger transcriptional modulation in UV-treated sam-ples, followed by samples treated with g radiation,while oxidative stress induced a considerably loweramount of gene expression changes (Figure 1B ofWeb supplement). A complete list of differentiallyexpressed genes after all the three treatments isshown in Table 1.

Validation of Microarray Results by

Quantitative Real-Time RT–PCR

In order to validate gene expression changesdetected by cDNA microarray, we utilized quantita-tive real-time RT–PCR. We performed real-time RT–PCR on a subset of genes that we detected as

differentially expressed in our microarray experi-ments. A scatter plot comparing gene expressionlevels measured bymicroarray and quantitative real-time RT–PCR is shown in Figure 2. Both methodsresulted in similar fold induction and repression ofdifferentially expressed genes (r¼ 0.8).

Identification of Common and Distinct Gene Expression

Patterns After Oxidative Stress, g Radiation, and UVRadiation in Human Dermal Diploid Fibroblasts

In order to better understand cellular processes inresponse to DNA damage after oxidative stress, gradiation, and UV radiation, we next examined geneexpressionpatterns after exposure toeach treatment,in order to identify discriminative genes that couldserve as a profile response to the different treatments.To extract themost discriminatory genes among thethree treatment groups, we utilized two differentstatistical approaches. The first one was an unsuper-vised approach based on genes identified as beingdifferentially expressed after treatment. The secondone was a supervised approach based on all 1901genes on the chip utilizing a Genetic Algorithm/KNearest Neighbor (GA/KNN) analysis [22].Based on the unsupervised hierarchical cluster of

genes that were identified as differentially expressedafter oxidative stress, g radiation, and/or UV radia-tion (Figure 2ofWeb supplement), all theUV-treatedsamples appeared to be most similar to one another(r¼0.772). The g-irradiated samples also appearedquite similar to one another (r¼0.712). Samplestreatedwith oxidative stress showed less similarity toone another and to the other treatments.Relationships between single members of treat-

ment groups were visualized in three-dimensionalspace using a principal component analysis (PCA)(Figure 3A). Within this analysis, Principal Compo-nent 1 (PC#1) was able to separate UV-treatedsamples from samples treated with oxidative stressor g radiation, while Principal Component 2 (PC#2)separated samples treated with oxidative stress fromthose treated with g radiation. An effort to exploregenes contributing most to the observed PCA separ-ation, which are therefore the most discriminatorygenes among the single treatment groups, resultedin the discovery of distinct gene groups that aremodulated in their transcription by the chosentreatments. These are shown in Figure 3B and listedin Table 2A and B.The second statistical approach we used to deter-

mine the most discriminating genes among samplestreatedwith eitheroxidative stress, g radiation, orUVradiation is based on a supervised pattern recogni-tion method called GA/KNN [22,23]. Within thisanalysis, theprogramwas suppliedwith the informa-tion on which samples belong to which treatmentgroup, and it was asked to extract, from among all1901 genes on the chip, the 100most discriminatorygenes that could separate the three treatment groups.

Figure 1. Cyclin E–associated kinase activity is inhibited after t-butyl-OOH, g radiation, and UV radiation. Exponentially growingcells were treated with 75 mM of t-butyl-OOH, 5 Gy g radiation,or 7.5 J/m2 UV radiation and harvested after 3 and 6 h. CyclinE–associated kinase activity was measured. Data are presented aspercent of activity in mock-treated controls and represent threeindependent experiments.

GENE EXPRESSION PROFILES AFTER DNA DAMAGE 69

Table 1. Genes Differentially Expressed After Oxidative Stress, g Radiation, and UV Radiation

IMAGE clone ID g Radiation t-Butyl-OOH UV Radiation

DNA replication/repair/cell-cycle regulationCyclin A2 32811 0.86 1.05 1.18Cyclin E1 357807 1.05 1.04 0.77FEN1 49950 0.77 1.03 1.38RFC3 (38 kDa) 256260 0.91 1.03 0.95RFC4 (37 kDa) 196676 0.75 1.00 1.20DDB2 209321 1.63 1.04 1.23p21 268652 2.64 1.38 3.16GADD45 310141 1.15 1.06 1.77Lamin B receptor 307225 0.83 0.99 1.03Lamin B2 485332 0.84 0.97 0.87Polymerase e 321207 0.92 1.00 0.90Cyclin G1 321743 1.51 1.08 1.33Cytokine-inducible kinase 324917 1.41 1.14 1.26PCNA 344109 1.38 1.12 1.89MCM7 530696 0.81 1.27 1.36MCM3 346838 0.77 0.98 0.78MCM2 358858 0.84 0.99 0.97Replication protein A2 366351 0.84 0.96 1.09PRIM2A 292326 0.99 0.83 0.58DOC-1R 485675 1.02 0.97 1.32

MitosisMAD2 129140 0.75 0.95 1.08NEK2 415089 0.68 1.16 0.93Aurora2 415639 0.76 0.96 0.89CDC20 563809 0.64 0.99 0.84

MAPK pathwayRibosomal protein S6 kinase, 90 kDa,

polypeptide 222711 1.01 0.96 0.74

Erythropoietin receptor 50015 1.03 0.98 0.79Ribosomal protein S6 kinase, 90 kDa,

polypeptide 3270209 0.97 0.95 0.77

MAP3K7IP1 470185 1.07 0.98 0.81MAP2K1 486638 1.04 1.01 1.33DUSP14 488150 1.08 1.04 1.27PAK4 128368 0.93 1.03 0.76

ApoptosisApo1 151767 1.49 1.09 1.28TRAF2 510375 0.92 1.04 0.83PTPN13 487097 0.87 1.06 0.86

TranscriptionSignal transducer and activator of

transcription 5A264200 1.09 0.79 0.97

Cyclin-dependent kinase 8 27944 0.95 1.02 0.63Nuclear factor kappa B-1 (NF-kB1) 114648 0.99 1.04 0.78TAF9 469931 0.99 1.01 1.46

Protein synthesis/degradation/targetingHeat shock 70 kDa protein 5 366350 1.11 1.31 1.39Heat shock 70 kDa protein 2 129370 0.96 1.00 1.36Serine (or cysteine) proteinase inhibitor,

clade H (hsp 47)563312 1.01 1.10 0.99

Group-specific component (vit D binding protein) 122140 1.17 1.32 0.73EIF4G3 270483 1.04 0.98 0.77Splicing factor, arginine/serine-rich 5 306176 1.15 0.99 1.39Eukaryotic translation initiation factor 4A,

isoform 2307532 1.21 1.08 1.52

Protein kinase H11 359191 1.11 1.02 1.34SFRS7 487937 0.90 0.97 1.23PRKCM 491336 0.83 0.92 0.83

70 HEINLOTH ET AL.

Table 1. (Continued )

IMAGE clone ID g Radiation t-Butyl-OOH UV Radiation

Transmembrane receptor/extracellular matrixTransmembrane 4 superfamily member 1 840567 0.99 1.23 1.48Insulin-like growth factor receptor 148379 1.08 1.06 0.70Insulin-like growth factor 1 144224 1.09 1.13 0.70Chemokine (C-X-C motif) receptor 4 144944 1.17 1.22 0.64Platelet-derived growth factor receptor

alpha (PDGFRA)489395 0.95 1.02 0.81

Cell growth and differentiationMCL1 145093 1.13 1.09 1.46PTK2 195731 1.03 0.91 0.83ACVR1 364929 1.00 0.96 0.75MAL 365360 1.09 1.06 0.75MACMARCKS 377132 1.00 1.03 1.37COL14A1 429561 1.08 4.56 0.62

GTP-binding proteinsBAIAP2 154655 1.10 1.05 0.73EDG1 307325 1.04 1.00 1.59GRK5 487709 1.04 0.94 0.66

MiscellaneousSLC9A1 67350 1.32 1.14 1.00Diacylglycerol kinase a (80 kDa) 366944 1.22 1.09 0.84ITGA2 525246 1.20 1.34 1.01Hyaluronan-mediated motility receptor 252202 0.95 1.12 0.97DDX18 33660 1.07 1.00 1.07NKG7 71606 1.22 1.18 0.71PPP1R1A 82526 1.051 1.04 0.74Sarcoma amplified sequence 121701 0.99 1.09 0.79Neuregulin 155716 1.24 0.90 0.67BCR 204681 1.13 0.94 0.76DPP4 208960 1.26 0.83 0.73NRL 219948 1.14 1.16 0.76PTP4A1 297595 1.31 1.09 1.60NR4A1 309893 1.00 1.07 1.70NR1H3 357775 1.14 0.98 0.63RBPMS 343443 1.02 1.50 0.75HYAL1 356890 1.02 0.96 0.76ARL1 469688 1.19 1.14 1.27CD59 486480 1.05 0.96 0.83PTPN9 488664 0.96 0.99 0.74

UnknownEST 209683 1.35 1.16 1.27EST 530912 0.79 1.08 1.16Matrix Gla protein 548131 1.27 0.83 1.25EST 48425 1.20 1.29 1.77EST, highly similar to I65981 fatty acid

omega-hydroxylase110844 0.98 0.96 0.63

EST 137959 1.06 0.90 1.36v-yes-1 Yamaguchi sarcoma viral related

oncogene homolog193913 1.00 0.88 0.63

EST, weakly similar to I38556 ribosomalprotein S6 kinase 2

211202 1.16 1.05 0.59

EST 233194 1.03 0.94 0.60Homo sapiens clone 25038 mRNA sequence 470073 0.99 1.02 0.74EST 471110 1.03 0.97 1.24DnaJ (heat shock protein 40 [Hsp40])

homolog, subfamily B, member 4546600 1.59 1.39 2.27

IMAGE, integrated molecular analysis of genomes and their expression; DDB2, damage-specific DNA binding protein 2; GADD45, growtharrest and DNA-damage-inducible; ID, identification.Ninety-four genes were identified as being differentially expressed after oxidative stress, g radiation, and/or UV radiation by utilizing IPLab2.0 and the mixed linear model analysis. These genes could be categorized in 11 different functional groups. The numbers represent thefold change of each gene as an average of all three cell cultures after one particular treatment. Bold numbers indicate in which treatmentgroup the gene was identified as being differentially expressed.

GENE EXPRESSION PROFILES AFTER DNA DAMAGE 71

The 100 genes selected as a result of this supervisedapproach and the associated expression ratios ofthose genes were subjected to two-dimensionalhierarchical cluster analysis (Figure 3 of Web sup-plement). Within this cluster, similar to the unsu-pervised hierarchical cluster, UV-treated samplesappeared to be most similar to one another(r¼0.79), followed by g-irradiated samples(r¼0.77), while samples treatedwith oxidative stresswere most dissimilar to one another (r¼0.41).This set of 100 most discriminatory genes was

introduced into PCA, and the separation in threedimensional space is shown in Figure 4A.Here again,

PC#1 was able to separate UV-irradiated samplesfrom samples treated with oxidative stress or g radia-tion, while PC#2 separated samples treated withoxidative stress from those treated with g radiation.The genes which contributedmost to this separationare shown in Figure 4 (B and C) and listed in Table 3(A and B).

Significant Differences Between Single Individualsin Their Response to DNA Damage

The degree of similarity between individuals intheir response to oxidative stress, g radiation, andUV radiation was analyzed by pairwise correlation

Figure 2. Levels of gene expression changes detected by micro-array analysis and quantitative real-time RT–PCR correlate well. Weperformed quantitative real-time RT–PCR testing for p21, RFC4,CDC20, and heat shock protein A5 in all the three human diploidfibroblast cultures after oxidative stress, g radiation, and UV radiation.Gene expression measurements were normalized to GAPDH expres-

sion in the single sample. Data are presented as a comparison oflevels of gene expression changes, detected by cDNA microarray andquantitative real-time RT–PCR, with the latter performed in tripli-cate. Correlation coefficient between quantitative real-time RT–PCRand cDNA microarray was r¼ 0.8.

72 HEINLOTH ET AL.

analysis (Figure 4A and B of Web supplement). Thethree human dermal diploid fibroblast cell culturesthat we used in this study showed significant inter-individual differences in their damage responses,which appeared to be treatment dependent. While gradiation seemed to evoke themost similar responsein all three individuals, with an average correlationcoefficient of 0.85 by single comparisons with oneanother, oxidative stress and UV radiation showedgreater inter-individual differences. The three cul-tures displayed an average inter-individual correla-tion coefficient in their gene expression changes of0.67 after UV radiation, whereas their correlationcoefficient after oxidative stress was in average 0.55.

DISCUSSION

Oxidative stress, g irradiation, and UV irradiationcan give rise to DNA damage and, as a response tothat, cell-cycle checkpoint activation. Checkpoint-enforced cell-cycle delays allow for DNA damage to

be repaired or, if repair is not possible, initiation ofcell death to exclude cells with a damaged genomefrom continued proliferation. Understanding theseevents is of major importance for comprehension ofthe etiology and development of therapies for dis-eases like cancer, which can arise, at least in part,due to a failure of cells to respond adequately toexposure to DNA-damaging agents. Multiple studieshave examined these processes in detail, describingprotein-protein interactions and signal transductioncascades.Wewere interested in comparing the cellu-lar gene expression responses to oxidative stress, girradiation, and UV irradiation. We were able todemonstrate both new and previously identifiedgene expression changes after oxidative stress, gradiation, and UV irradiation. The single treatmentsresulted in very distinct gene expression modula-tions that could discriminate between them. Thesegene expression patterns allow the associationof specialized signaling pathways to the different

Figure 3. Principal component analysis of differentially expressedgenes after t-butyl-OOH, g irradiation, and UV irradiation identifiedwith IPLab 2.0 and mixed linear model analysis. (A) PCA based on 95genes which where identified as being differentially expressed inresponse to t-butyl-OOH, g radiation, and/or UV radiation. Cellcultures and treatments are indicated in the figure. The axes arelabeled according to the percent of variation in the data eachprincipal component captures. (B) Clustering tree of most discrimi-natory genes among samples treated with t-butyl-OOH, g radiation,

and UV radiation within PCA using the average log2 calibrated ratiofor all 36 hybridizations in Eisen’s algorithm [49]. PC#1 contributorsdiscriminated between UV-irradiated samples and samples treatedwith t-butyl-OOH or g radiation. PC#2 contributors discriminatedbetween samples treated with t-butyl-OOH and g radiation. Samplecodes are the treatment identifiers. Shade of color in the cluster heatmap represents the extent of differential expression: red, induced;green, repressed; black, unchanged. The branch length of the den-drogram denotes the amount of similarity between clusters.

GENE EXPRESSION PROFILES AFTER DNA DAMAGE 73

treatments we used andmay aid in the identificationof biomarkers for exposures to specific DNA-dama-ging agents.Gamma radiation had the clearest influence on

different cell-cycle phases, specifically the S- and M-phases. Preparation for S-phase, in which DNAreplication takes place, is initiated in late M-/earlyG1-phase, when cell division cycle (CDC) 6 and theminichromosome maintenance (MCM) proteins(MCM2-7) start to assemble at the origin of replica-tion and form the prereplication complexes. At theG1/S-transition CDC45 is loaded, along with otherproteins, on the now-forming preinitiation com-plexes. In S-phase,CDC6 is phosphorylated by cyclinA/cyclin dependent kinase (CDK) 2 complexes [24],which leads to its dissociation and ubiquitin-medi-ated degradation. This event releases the replicationcomplex and starts DNA replication. Within theelongation process, the MCM proteins play a majorrole [25]. The six family members form two func-tionally distinct subgroups: MCM4/6/7 form thecatalytic core with helicase activity [26], whileMCM2/3/5 have a regulatory function. The MCM4/6/7 complex is inactivated after phosphorylation of

MCM4 by cyclin A/CDK2 complexes, resulting inreplication stopping [27]. Replication of single-stranded DNA at the replication fork requires RNAprimers, which are synthesized by DNA polymerasea-primase. On the leading strand, the elongationprocess starts from a single primer, while the laggingstrand is replicated from numerous primers at eachOkazaki fragment. Primer elongation starts with thebinding of the multisubunit replication factor C(RFC), which then recruits proliferating cell nuclearantigen (PCNA), anchoringDNApolymerase dor e tothe DNA. During DNA-elongation, lamin A and B,located between the inner nuclear membrane andperipheral chromatin, co-localize to PCNA and RFC[28]. Removal of the RNA-primers by RNase H1 isaccomplished by flap structure-specific endonu-clease (FEN1) and the resulting gap is filled in byDNA polymerase and joined by DNA ligase 1. (Forreviews see [29–31]).Thegenes involved in theseprocesses,FEN1,RFC3,

and RFC4, lamin B2 and lamin B receptor, polymer-ase e, replication protein A2 and several MCMs(MCM2, 3, and 7), were all downregulated after girradiation (indicated in green in Figure 5). The

Figure 3. (Continued )

74 HEINLOTH ET AL.

expression levels of these genes were not signifi-cantly changed after oxidative stress or UV irradi-ation, with the exception of FEN1, which wasupregulated after UV treatment, and DNA-primase,which was downregulated after UV irradiation. The

only gene out of this subgroup of genes involved inDNA replication that was upregulated in both UV-and g-irradiated samples, was PCNA. This gene has avariety of functions within the cell, including itsinvolvement in DNA replication and repair and

Table 2A. Most Discriminatory Genes Within PC#1 That Separate Samples Treated With UV Radiation From ThoseExposed to Either g Radiation or t-Butyl-OOH

IMAGE clone ID Unigene descriptionGene

symbol PC#1 score Gene function

148379 Insulin-like growth factor 1 receptor IGF1R 0.12558 Transmembrane receptor292326 Primase, polypeptide 2A PRIM2A 0.12509 DNA replication364929 Activin A receptor, type 1 ACVR1 0.12338 Cell growth and

differentiation154655 BAI 1-associated protein 2 BAIAP2 0.1233 Cell growth and

differentiation29682 EST EST 0.12174 Unknown114648 Nuclear factor of kappa light

polypeptide gene enhancerin B-cells 1 (p105)

NF-jB 0.1216 Transcription

489395 Platelet-derived growth factorreceptor, alpha polypeptide

PDGFRA 0.12059 Transmembranereceptor

129370 Heat shock 70 kDa protein 2 HSPA2 � 0.10732 Stress response307325 Endothelial differentiation, sphingolipid

G-protein-coupled receptor 1EDG1 � 0.1109 Transmembrane receptor

486638 Dishevelled, dsh homolog 3 (Drosophila) MAP2K1 � 0.1113 MAPK pathway310141 Growth arrest and DNA-damage-inducible,

alphaGADD45 � 0.11209 Cell-cycle regulation

429622 POU domain, class 2 transcription factor 2 POU2F2 � 0.11313 Transcription309893 Nuclear receptor subfamily 4,

group A, member 1NR4A1 �0.12173 Nuclear receptor

469931 TAF9 RNA polymerase II, TATA box bindingprotein (TBP)-associated factor, 32 kDa

TAF9 � 0.12367 Transcription

Analysis based on genes identified as being differentially expressed after treatment with t-butyl-OOH, g radiation, or UV radiation byIPLab 2.0 or mixed linear model analysis. Bold indicates genes that were parallelly identified to belong to the group of most discriminatorygenes by GA/KNN.

Table 2B. Most Discriminatory Genes Within PC#2 That Separate Samples Treated With g Radiation From Those Exposedto t-Butyl-OOH

IMAGE clone ID Unigene description Gene symbol PC#2 score Gene function

209321 Damage-specific DNA bindingprotein

DDB2 0.19779 Cell-cycleregulation

268652 Cyclin-dependent kinase inhibitor 1A(p21, Cip1)

p21 0.17373 Cell-cycle regulation

264200 EST EST 0.15773 Unknown548131 EST EST 0.15616 Unknown488150 Dual specificity phosphatase 14 DUSP14 0.15412 MAPK pathway563809 CDC20 homolog (S. cerevisiae) CDC20 �0.1352 Mitosis488092 EST EST � 0.13592 Unknown307225 Lamin B receptor LBR � 0.13741 DNA-replication485332 Lamin B2 LAMB2 � 0.14062 DNA-replication415639 Serine/threonine kinase 15 STK15 (Aurora2) �0.14646 Mitosis530696 MCM7 deficient 7 (S. cerevisiae) MCM7 � 0.15153 DNA-replication346838 MCM3 deficient 3 (S. cerevisiae) MCM3 � 0.15643 DNA-replication415089 NIMA-related kinase 2 NEK2 �0.17277 Mitosis

Analysis based on genes identified as being differentially expressed after treatment with t-butyl-OOH, g radiation, or UV radiation byIPLab 2.0 or mixed linear model analysis. Bold indicates genes that were parallelly identified to belong to the group of most discriminatorygenes by GA/KNN. NIMA, never in mitosis gene a.

GENE EXPRESSION PROFILES AFTER DNA DAMAGE 75

Figure 4.

76 HEINLOTH ET AL.

interaction with a large number of other geneproducts. PCNA is regulated transcriptionally andpost-transcriptionally in a p53-dependent manner;in cellswith lowp53 levels, thepresenceof PCNAwilllead to DNA replication, while in cells with high p53levels,DNA repair is initiated [for reviewRef. 32]. Theinteractionofp53withPCNA influences thepartnersof PCNA in both processes. One of these partners isFEN1, which interacts in DNA replication [33] andBER with PCNA [34]. While it was downregulatedafter g irradiation in our experiments, FEN1 wasupregulated after UV irradiation. This is in congru-ence with studies which observed increased sensi-tivity against UV light exposure after disruption ofthe BER mechanism [35]. As illustrated in Figure 5,both g and UV irradiation induced in additionseveral p53 and/or E2F-dependent gene expressionchanges.

Another major subgroup of genes modulated afterg irradiation was involved in mitosis. Mitotic arrestdeficient 2 (MAD2), NIMA-related kinase 2 (NEK2),Aurora2, and CDC20 belong to this group, all ofthese were downregulated after g irradiation but notsignificantly changed after oxidative stress or UVirradiation. MAD2 is an inhibitor of the metaphase-anaphase transition in response to misalignment ofchromosomes [36]. MAD2 inhibits CDC20 [37],which is required for the activation of the Ana-phase-Promoting-Complex [38]. NEK2 localizes tothe centrosome and is important for its integrity,playing an important role in regulating centrosomeseparation. Overexpression of NEK2 induces split-ting of centrosomes [39]. Aurora2 is a centrosome-associated kinase [40,41] and is important for align-ment of chromosomes on the metaphase plate [42].The downregulation of all of these genes suggests

Table 3A. Most Discriminatory Genes Within PC#1 That Separate Samples Treated With UV Radiation From ThoseExposed to Either g Radiation or t-Butyl-OOH

IMAGEclone ID Unigene description Gene symbol PC#1 score Gene function

322710 Bone morphogenic protein receptor, type 1A BMPR1A 0.13528 Transmembranereceptor

114648 Nuclear factor of kappa lightpolypeptide gene enhancerin B-cells 1 (p105)

NF-jB 0.13527 Transcription

489395 Platelet-derived growth factorreceptor, alpha polypeptide

PDGFRA 0.13232 Transmembranereceptor

377181 Cas-Br-M (murine) ectropic retroviraltransforming sequence b

CBLB 0.13158 Immune function

563395 Homeo box C10 HOXC10 0.13134 Development148379 Insulin-like growth factor 1 receptor IGF1R 0.13002 Transmembrane

receptor429312 Ribosomal protein S6 kinase, 52 kDa,

polypeptide 1RPS6KC1 0.12961 Unknown

486055 Cytochrome p450, subfamily 1(dioxin-inducible, polypeptide 1(glaucoma 3, primary infantile)

CYP1B1 � 0.10298 Metabolism

344109 Proliferating cell nuclear antigen PCNA � 0.10464 DNA replication/repair137959 Hepatocellular carcinoma-associated

protein HCA4HCA4 � 0.1053 Unknown

470792 General transcription factor IIB GTF2B � 0.10675 Transcription49950 Flap structure-specific endonuclease 1 FEN1 � 0.11135 DNA replication/repair546600 DnaJ (Hsp40) homolog, subfamily B, member 4 DNAJB4 � 0.11573 Stress response309893 Nuclear receptor subfamily 4,

group A, member 1NR4A1 �0.12587 Nuclear receptor

Analysis based on genes identified as being most discriminatory between samples treated with t-butyl-OOH, g radiation, or UV radiationby GA/KNN. Bold indicates genes that were parallelly identified to belong to the group of most discriminatory genes by unsupervisedanalysis with IPLab 2.0 or mixed linear model analysis.

Figure 4. Principal component analysis of the most discriminatorygenes between samples treated with t-butyl-OOH, g radiation, andUV radiation identified with GA/KNN. (A) PCA based on the 100genes which were identified as the most discriminatory betweensamples treated with t-butyl-OOH, g radiation, and UV radiation. Cellcultures and treatments are indicated in the figure. Axes are labeledas described in Figure 3A. (B) Clustering tree of the most discri-minatory genes between samples treated with t-butyl-OOH, g

radiation, and UV radiation within PCA. PC#1 contributors dis-criminated between UV-irradiated samples and samples treated witht-butyl-OOH or g radiation. PC#2 contributors discriminated be-tween samples treated with t-butyl-OOH and g radiation. Genenumbers are integrated molecular analysis of genomes and theirexpression (IMAGE) clone identifiers [15]. Features of the cluster treeand heat map are described in Figure 3B.

GENE EXPRESSION PROFILES AFTER DNA DAMAGE 77

that, in response to exposure to g radiation, the cellexerts a significant effort to prevent progressionthrough mitosis, at least until the resulting DNAdamage can be repaired.To focus furtheron the strongeffect of g irradiation

on the regulation of genes involved in cell-cycleregulation, DNA replication, and DNA repair,we selected genes involved in these processes thatare represented on themicroarray chips we used andperformed a hierarchical cluster analysis with thissubset of genes across all treatment groups. The resultof this analysis is shown inFigure 6. As expected fromour former analyses, this selection of genes and theirexpression pattern after g irradiation resulted in amost similar behavior of the cell cultures treatedwithg radiation, while oxidative stress–treated samplesand UV-irradiated samples were not as well coordi-nated in the modulation of their expression of thisparticular subset of genes. In addition to the genesalready described, CDC25B, a regulator of the G2/Mtransition, appeared to be specifically downregulat-ed only after g irradiation. It is apparent that geneexpression changes after g irradiation strongly in-fluenced specific functional families. It will beinteresting to see if other genes that we identifiedto be differentially expressed after g irradiation havestill unknown implications in DNA replication,repair, or regulation of S-phase progression or prog-ression through mitosis.One aimof this studywas todeterminewhichgene

expression changes after g irradiation were induced

by oxidative stress, which is a subcomponent of theintracellular modifications caused by g irradiation.While comparing gene expression changes afteroxidative stress, g radiation, and UV irradiation,changes after oxidative stress and g irradiation ap-peared to be more similar to one another than tochanges in response to UV radiation (Figure 3A,B).Nevertheless, we were not able to identify a subset ofgenes that were specifically changed after oxidativestress and g irradiation. This could be due to severalreasons. It appeared that within our experimentaldesign, checkpoint induction and DNA-damageresponse after oxidative stress were not connectedwith a large number of significant gene expressionchanges. We observed p21 upregulation, whichreinforced that our treatment resulted in checkpointinduction.Our laboratoryshowedrecentlythat treat-ment with 75 mM of t-butyl-OOH induces heme-oxygenase-1 protein expression after 4 h in humandermal fibroblasts [43]. This is seen as evidencethat our treatment are in fact generating cellularresponses to damage generated by ROS, because ofthe induction of heme-oxygenase-1 by peroxidesin human dermal fibroblasts [44]. However, in thisstudy heme-oxygenase-1 was not found to bestrongly induced by t-butyl-OOH on the mRNAlevel. One possible explanation for the limitednumberof gene transcriptionchanges afteroxidativestress could be different kinetics of checkpointresponses observed after oxidative stress in contrastto g andUV irradiation.Comparisonof the reduction

Table 3B. Most Discriminatory Genes Within PC#2 That Separate Samples Treated With g Radiation From Those Exposedto t-Butyl-OOH

IMAGEclone ID Unigene description Gene symbol PC#1 score Gene function

264200 Signal transducer and activator oftranscription 5A

STAT5A 0.19172 Transcription

209321 Damage-specific DNA bindingprotein 2 (48 kDa)

DDB2 0.18199 Cell-cycleregulation

132588 Hypothetical protein FLJ13433 EST 0.16184 Unknown328284 Islet amyloid polypeptide IAPP 0.16115 Pancreatic islet

function151767 Tumor necrosis factor receptor

superfamily, member 6TNFRSF6 0.16083 Apoptosis

548131 Homo sapiens calmodulin-I (CALM1)mTNA, 30UTR, partial sequence

0.15902 Intracellular calciumhomeostasis

470683 Beta-amyloid binding protein precursor BBP 0.15806 Apoptosis488989 B-cell CLL/lymphoma 7B BCL7B � 0.16399 Unknown366384 Myosin binding protein H MYBPH � 0.16447 Cellular movement625871 Thioredoxin reductase 1 TXNRD1 � 0.1686 Stress response415639 Serine/threonine kinase 15 STK15 (Aurora2) �0.17133 Mitosis415089 NIMA-related kinase 2 NEK2 �0.17391 Mitosis563809 CDC20 homolog (S. cerevisiae) CDC20 �0.17709 Mitosis484963 Metallothionein 2A MT2A �0.17713 Cell proliferation

Analysis based on genes identified as being most discriminatory between samples treated with t-Butyl-OOH, g radiation, or UV radiationby GA/KNN. Bold indicates genes that were parallelly identified to belong to the group of most discriminatory genes by unsupervisedanalysis with IPLab 2.0 or mixed linear model analysis.

78 HEINLOTH ET AL.

of cyclin E–associated kinase activity after oxidativestress, gandUVradiationdisplayeda slower responseafter oxidative stress.While the two latter treatmentsresulted in a significant reduction already 3 h aftertreatment, kinase activity after oxidative stress wasnot reduced to similar levels until 6hafter treatment.To examine whether gene expression changes aredelayed in a similar manner, we performed micro-array analysis onacell culture treatedwith75mMof t-butyl-OOH, and harvested after 9 h instead of 6 h.Hybridization of the prepared mRNA and analysisresulted in fewer, instead of more, gene expressionchanges (data not shown), providing evidenceagainst a delayed kinetic of gene expression changesafter oxidative stress. This led us to speculate that inthe case of oxidative stress, mainly post-transcrip-tional modifications, like phosphorylation events,are activated and are sufficient for a checkpointinduction and damage response. While our analysisfocusesontranscriptionalchanges,proteomicappro-aches will be necessary to elucidate other responsesto oxidative stress. In addition, our custom-builtmicroarray chip represents only a limited subset ofgenes from the human genome. Although several

known oxidative response genes are represented, itcould be that amajor pathway activated by oxidativestress might not be represented. It is clear that dose-dependent changes can occur and this will requiremore studies with additional doses.UV radiation had the highest impact on transcrip-

tion in our study. One pathway that was very signi-ficantly modulated by UV radiation while notaffected by oxidative stress or g radiation is theMAPKpathway (Figure 5). This is in congruencewithalreadypublisheddata [45–47].While former studiesdemonstrate activationof theMAPKpathwaysbyUVradiation, we have evidence for a transcriptionalmodulation of genes involved in these pathwaysafter UV radiation, but not after oxidative stress or gradiation. Two other subgroups of genes that weremainly modified by UV irradiation but not influ-enced by oxidative stress or g irradiation are genesinvolved inprotein synthesis/degradationand in cellgrowth and differentiation.Within our experimental setting, more genes were

downregulated than upregulated after UV irradia-tion. The question remains if this effect is due to ageneral block of transcription after UV irradiation or

Figure 5. Model for regulation of different gene expressionpatterns after g and UV radiation. While only g radiation influencesthe differential expression of genes involved in mitosis, both g andUV radiation modulate the expression of p53 and E2F-regulatedgenes, although with different expression patterns. Genes involved

in MAPK pathway and protein-synthesis and degradation aredifferentially expressed only after UV radiation. Green indicatesdownregulation, red signifies upregulation, and genes shown inyellow were not identified as being differentially expressed withinour analyses.

GENE EXPRESSION PROFILES AFTER DNA DAMAGE 79

to specific gene expression responses. A recent reportfrom Mone et al. [48] describes local inhibition oftranscription after UV radiation (UVC) in primaryfibroblast cells. Using a comparable cell culturemodel, the authors had to apply doses of UV ir-radiation that far exceeded ours to see this effect(30–50 J/m2 instead of 7.5 J/m2 in our study).Another argument against a general block of tran-scription in our system is the fact that we saw notonly downregulated genes, but also a variety of up-regulated genes, after UV radiation.Our present study demonstrates that comparable

dosages of oxidative stress, g radiation, and UV radi-ation resulted in distinct gene expression changes,withmajor impact onDNA transcription andmitosisin the case of g radiation, while the MAPK pathway,protein synthesis, and cell growth and differentia-tion were affected by UV radiation. In order toidentify a subset of genes that would allow discrimi-nation of the transcriptional responses to all threetreatments, we analyzed the dataset with twodifferentmethods to extract themost discriminatorygenes: PCA of differentially expressed genes and GA/

KNN. Although both statistical tools are indepen-dent from one another and utilize different gene sets(we used only differentially expressed genes (PCA) incontrast to all the genes on the chip (GA/KNN)), asubset of discriminatory geneswas identified bybothmethods. The genes that were identified uniquely byone of the two approaches belong mainly to similarfunctional classes. These results can be the basisfor thedevelopmentof simple screeningmethods forbiomarkers indicating exposures to different types ofDNA-damaging agents, especially as we assured withour experimental setting that the discovered geneexpression changes were reproducible in differentindividuals with different genetic backgrounds.

ACKNOWLEDGMENTS

We thank Jeffrey Tucker, Danica Ducharme, andJonathan Miller for excellent technical support, andJeffrey Chou, Alex Merrick, Theodora Devereux,and Carol Trempus for helpful discussions andcritical review of the manuscript. A.N.H. was sup-ported by DFG grant no HE 3255/1-1.

Figure 6. Hierarchical cluster analysis of DNA replication/repair and cell-cycle genes in samples treated witht-butyl-OOH, g radiation, and UV radiation. Cluster analysis of all DNA replication/repair and cell-cycle genesrepresented on NIEHS ToxChip 2.0 using the average log2 calibrated ratio for all 36 hybridizations in Eisen’salgorithm [49]. Gene numbers are IMAGE clone identifiers [15]. Features of the cluster tree and heat map aredescribed in Figure 3B.

80 HEINLOTH ET AL.

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