SHORT REPORT

Fluorescent cDNA microarray hybridization reveals complexity andheterogeneity of cellular genotoxic stress responses

Sally A Amundson*,1, Mike Bittner2, Yidong Chen2, Je�rey Trent2, Paul Meltzer2 andAlbert J Fornace Jr1

1National Institutes of Health, National Cancer Institute, Bethesda, Maryland, MD 20892, USA; 2National Human GenomeResearch Institute, Bethesda, Maryland MD 20892, USA

The fate of cells exposed to ionizing radiation (IR) maydepend greatly on changes in gene expression, so that animproved view of gene induction pro®les is important forunderstanding mechanisms of checkpoint control, repairand cell death following such exposures. We have used aquantitative ¯uorescent cDNA microarray hybridizationapproach to identify genes regulated in response to g-irradiation in the p53 wild-type ML-1 human myeloidcell line. Hybridization of the array to ¯uorescently-labeled RNA from treated and untreated cells wasfollowed by computer analysis to derive relative changesin expression levels of the genes present in the array,which agreed well with actual quantitative changes inexpression. Forty-eight sequences, 30 not previouslyidenti®ed as IR-responsive, were signi®cantly regulatedby IR. Induction by IR and other stresses of a subset ofthese genes, including the previously characterized CIP1/WAF1, MDM2 and BAX genes, as well as nine genesnot previously reported to be IR-responsive, wasexamined in a panel of 12 human cell lines. Responsesvaried widely in cell lines with di�erent tissues of originand di�erent genetic backgrounds, highlighting theimportance of cellular context to genotoxic stressresponses. Two of the newly identi®ed IR-responsivegenes, FRA-1 and ATF3, showed a p53-associatedcomponent to their IR-induction, and this was con®rmedboth in isogenic human cell lines and in mouse thymus.The majority of the IR-responsive genes, however,showed no indication of p53-dependent regulation,representing a potentially important class of stress-responsive genes in leukemic cells.

Keywords: cDNA microarray; ionizing radiation;leukemia; p53; genotoxic stress

Genotoxic stresses, such as ionizing radiation (IR), canelicit a wide variety of cellular responses, from cell-cycle arrest to mutation, transformation, or cell death.On the molecular level, early responses to IR includeactivation of proteins such as p53 and NFkB, changesin protein localization, engagement of signal cascadesand transcriptional induction of speci®c genes such asCIP1/WAF1 and MDM2. Many factors, both cell-type

and stress-speci®c, can interact to determine the ®naloutcome for the cell. IR has proved a useful probe forthe study of basic processes such as cell-cycleregulation, apoptotic pathways, and DNA metabolismand the identi®cation of new IR-response genes mayalso provide novel targets for future experimentalapproaches in radiotherapy.

The human myeloid cell line ML-1 was selected forthis study based on our previous experience with stressgene induction in many di�erent cell lines. Variousfactors contribute to the IR-responsiveness of cell lines.For instance, the p53 wild-type status of ML-1 allowedthe detection of many genes, such as CIP1/WAF1,GADD45 and MDM2, which require p53 function foroptimal IR-induction. Importantly, this cell linecontains endogenous wild-type p53, so the resultsobtained represent cellular responses with physiologi-cal levels of p53, rather than the unnatural and oftenhighly overexpressed levels resulting from arti®ciallyengineered systems. In addition, the fact that ML-1 is amyeloid cell line, prone to undergo rapid apoptosisfollowing genotoxic stress, also results in the inductionof genes speci®cally associated with this process, suchas BAX, MCL1, GADD34 and BCL-XL (Zhan et al.,1994b, 1997). The genes we knew to be IR-regulated inML-1 prior to this study gave us a wide range of targetresponses we could expect to detect on the microarray,from 450-fold induction for CIP1/WAF1 to approxi-mately tenfold reduction for c-MYC.

Since the initial development of cDNA microarrayhybridization (Schena et al., 1995), there has beenconsiderable interest in this rapidly emerging technol-ogy. The ability to compare relative levels of thousandsof mRNA transcripts simultaneously in a singlehybridization has the potential to contribute greatlyto our understanding of many di�erent ®elds.Development of a quantitative approach for themeasurement of relative changes in gene expressionwould o�er the added advantage of expanding thisapproach beyond simple pair-wise comparisons.Demonstrated applications have included monitoringof di�erential gene expression in wild-type andtransgenic plants (Schena et al., 1995) and of a humanmelanoma cell line and its non-tumorigenic variant(DeRisi et al., 1996), expression of heat-shock genes ina human cell line (Schena et al., 1996), metabolic shiftin yeast (Lashkari et al., 1997), and the identi®cation ofgenes related to in¯ammatory disease (Heller et al.,1997).

Transcriptional stress-response is an extremelycomplex area, with the response of many genesdependent on intermeshing signal transduction activ-

*Correspondence: SA Amundson, NIH, National Cancer Institute,37 Convent Dr., Bldg. 37/rm. 5C09, Bethesda, Maryland, MD 20892,USAReceived 17 July 1998; revised 14 January 1999; accepted 14 January1999

Oncogene (1999) 18, 3666 ± 3672ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00

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ities, cell type speci®c factors and genetic background.This can make it di�cult to assess the importance ofthe response of a single gene on the basis of data fromone or a few cell lines or individuals. The applicationof a high-throughput screening method, such as cDNAmicroarray hybridization, may be necessary to developa clearer picture of stress-responsive pathways. Wehave extended the application of ¯uorescent cDNAmicroarray hybridization to the study of a complexstress-responsive system in a non-engineered humancell line.

The microarrays used in this study included ageneral sampling of human genes (622 ESTs) plusanother set of genes (616 ESTs) which were chosen onthe basis of their known roles in cancer or lymphoidbiology. A panel of housekeeping genes and otherinternal controls was also included on the array. AllESTs were selected from the UniGene database withthe assistance of G Schuler and M Boguski. Arepresentative quadrant of a hybridized microarray isshown in Figure 1. Induced transcripts hybridize withmore of the probe from the IR-treated sample (labeledin red), resulting in red spots, such as that seen at theCIP1/WAF1 target indicated in Figure 1. A transcriptdown-regulated by IR, such as c-MYC, would similarlyproduce a green spot (Figure 1). Intermediateinduction ratios result in a gradation of color, suchas MBP-1 in Figure 1.

Among the transcripts signi®cantly changed byradiation treatment (Table 1) were a number of genespreviously known to be radiation inducible, as well asmany genes not previously reported as ionizing-radiation regulated. Table 1 also gives the meanintensities of hybridization to the unirradiated controlon the microarray. This measure has previously beenshown to correlate roughly with transcript abundance(Schena et al., 1995, 1996), and demonstratesidenti®cation of IR-modulated genes over three ordersof magnitude of basal expression. It should be notedthat many of the stress regulated transcripts identi®edin Table 1 are known to be expressed at very low levelsin ML-1 cells, consistent with their relative hybridiza-tion intensities on the array. For example, GADD45and CIP1/WAF1 represent approximately 1/105 tran-scripts in unirradiated cells.

A subset of the IR-responsive genes indicated by themicroarray with a range of relative ratios were chosenfor further study. Probes were obtained from the sameplasmids used as targets on the array and g-rayinduction of these genes was con®rmed in independentexperiments by Northern blot hybridization (Figure 2).Estimates of induction or repression as measured bythe microarray were compared to quantitative hybridi-zation with single labeled probes. As indicated in Table1, estimated expression varied by less than twofold formany transcripts. As will be discussed in more detailelsewhere (Bittner et al., in preparation), induction insome cases diverged from that measured by microarrayhybridization in an apparently EST speci®c manner.However, it should be noted that all tested sequencesthat were identi®ed on the microarray as inducedshowed an appreciable (42-fold) induction by thequantitative hybridization approach, with the exceptionof MRC-OX, which showed only 1.5-fold induction. Inthe case of genes showing less than 2.4-fold inductionby the microarray, useful data may still be obtainable.

For example, MCL-1 showed 1.9-fold induction by themicroarray and 2.5-fold induction by quantitativehybridization. This gene has previously been shown

Figure 1 One quadrant of the microarray hybridized to RNAfrom untreated ML-1 cells (green ¯uorochrome) and ML-1 cells4 h after treatment with 20 Gy137 Cs g-rays (red ¯uorochrome).Targets appearing as yellow spots have equal representation ofboth ¯uorochromes and indicate no change in expression by theIR treatment. Red spots are targets increased by the treatment,and green targets are decreased. The horizontal row of green dots,`landing lights', at the top and bottom are DNA labeled prior toprinting on the array and serve as orientation markers for thecomputerized scanner. Representative target spots are identi®ed inthe enlarged segment. EST targets were prepared by PCRampli®cation and arrayed on poly-L-lysine coated glass slides byhigh speed robotic printing as previously described (DeRisi et al.,1996). A complex cDNA probe was prepared from whole-cellRNA by a single round of reverse transcription in the presence of¯uorescent dNTP (Cy3 dUTP or Cy5 dUTP, Amersham). Probeswere hybridized to the slides for 16 h in 36SSC at 658C in thepresence of blockers. Hybridized slides were washed at roomtemperature in 0.56SSC, 0.01% SDS, then in 0.066SSC. Thetwo ¯uorescent intensities were scanned separately using a laserconfocal microscope, and the DeArray program was then used toidentify target sites by image segmentation, calibrate relativeratios, and to develop con®dence intervals for testing thesigni®cance of the ratios obtained (Chen et al., 1997). Localbackground was calculated for each target location. A normal-ization factor was estimated from a set of 88 internal controltargets (DeRisi et al., 1996) with a theoretical ratio of 1.0, and thecon®dence interval for the array was estimated from the varianceof these 88 control ratios from the expected value of 1.0. Theratios for all the targets on the array were then calibrated usingthe normalization factor, and ratios outside the 99% con®denceinterval (50.54 or 42.37) were determined to be signi®cantlychanged by the radiation treatment

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to be IR-responsive in the ML-1 cell line (Zhan et al.,1997). The full listing of sequences used on thismicroarray and their relative ratios can be accessedvia the website at http://rex.nci.nih.gov/RESEARCH/basic/lbc/fornace.htm.

The timecourse of induction for nine of these geneswas examined in ML-1 cells. The response over time ofRag cohort 1 (RCH1) (Cuomo et al., 1994), a newlyrecognized IR-down-regulated gene, was very similar tothe response of TOPOII (data not shown). Both

repressed genes showed a similar rapid decrease ofmRNA levels following irradiation, and remainmaximally repressed 24 h after treatment. The levelsof most of the newly-identi®ed IR-induced genes roserapidly following treatment, peaked by 4 h, anddeclined again to near the original levels by 24 h aftertreatment (data not shown), following the pattern ofrapid response typical of many stress-induced immedi-ate-early genes (Fornace, 1992; Holbrook et al., 1996;Smith and Fornace, 1996a). By analogy, the genesdescribed here may have roles in acute cellularresponses to damage and may share some regulatorymechanisms with previously characterized IR-responsegenes.

Induction of nine of the newly-identi®ed stress-response genes was next measured in a panel ofhuman cancer cell lines to determine the scope of theirIR-response, and to monitor for induction byexposure to two DNA-base-damaging agents, thealkylating agent methyl methanesulfonate (MMS)and ultraviolet (UV) radiation. The cell lines used inthis comparison included six lines of myeloid-lymphoid lineage (ML-1 (myeloid), Molt4 (lym-phoid), SR (lymphoid), CCRF-CEM (lymphoid),HL60 (myeloid) and K562 (myeloid)) two lung cancerlines (A549 and H1299), two breast carcinoma lines(MCF7 and T47D) and the colon cancer line RKOalong with its derivative transfected with E6 (RKO/E6) (Zhan et al., 1993). A summary of these results isshown in Figure 3. Induction levels of MDM2, CIP1/WAF1 and BAX were also measured for comparison.These three genes are p53-regulated (Ko and Prives,1996); in the case of CIP1/WAF1, it is regulated byboth p53-dependent and independent mechanisms(Gorospe et al., 1996), while BAX induction by IRappears to require p53 plus an `apoptosis-pro®ciency'factor frequently present in cells that undergo rapidapoptosis after IR (Zhan et al., 1994b). As expected,these three genes were more responsive in the p53 wtlines with little or no IR-responsiveness in the p53de®cient lines. As reported previously (Zhan et al.,1994b), strong BAX induction was only seen inmyeloid-lymphoid lines with weaker induction inMCF-7 cells. In the case of the SR leukemia line,constitutive BAX expression was approximately anorder of magnitude greater than in the other lines and

Table 1 Stress gene responses in g-irradiated human myeloid cells

Transcript Image ID Microarraya

MeanGreen

Intensityb

CIP1/WAF1ATF3FASIAP-1RELBCYCLIN-IRABGADD45FRA1IL-8CSF-1BCL3MIP1ac-FOSJUN-BPC-1CDC5MRC OX-2ERR1MDM2Immunoglobulin J chainOX40 ligandDNA ligase IIIcytochrome p450 4AMEK1TPK receptor UFORetinoic acid gamma-1HPTP alphaMBP-1SSATBAKMBP-2iL-TMPMIP1betaN-RASnucleotide binding protein mRNACAP-RBCL-XL

ch-TOGERKCDC2SATB1SPI-BERF-1MADP2 homologneuron-speci®c protein geneTOPO IICDK RS2RCH-1c-MYC

26865242824815176712963252681512391525303101411105033286921245542364221533552647430947752753280376513634706021470751610233532647006212046648607411250047125248713029150341452

e

234642431432056332840312142048677

e

430604874173465342331942950934808526541280892480323591192811651699

42.0c

11.3c

9.59.3d

7.2d

6.96.45.8d

4.8c

4.84.74.6c

4.34.24.23.4c

3.43.4d

3.23.2d

3.13.13.03.02.92.82.82.82.8c

2.7c

2.7c

2.52.5d

2.52.52.52.42.3c

0.530.510.470.46c

0.450.440.430.380.34c

0.270.23c

0.12c

1121784135213210279741688223067253073565844751314189234312592904903283313106362181885285288282206541603414623833523413945773139209932319501303643747693520543809837173139352493217913

aRatios of relative induction by g-rays compared to basal levels inML-1 cells. See also http://rex.nci.gov/RESEARCH/basic/lbc/for-nace/htm. bFluorescence intensity of untreated control on themicroarray. cMicroarray measurement con®rmed by quantitativedot blot hybridization where expression varied by less thantwofold. (Bittner et al., in preparation). dQuantitation varied bymore than twofold (Bittner et al., in preparation). eInserts for BAK(Chittenden et al., 1995) and BCL-XL (Boise et al., 1993) from clonesother than Image Consortium ESTs

Figure 2 Representative Northern blot analysis con®rming genesshowing altered expression by the microarray following IRtreatment. C: untreated control, g: 4 h after g-irradiation.Molecular weights of hybridizing bands were consistent withpublished transcript sizes. ATF3 hybridized to two di�erent sizedbands, corresponding to the alternately spliced forms previouslyreported (Chen et al., 1994a)

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appreciable induction was not seen. In contrast tothese previously described genes, the ionizing radiationresponse of the newly characterized genes shows farmore heterogeneity among di�erent cell lines (Figure3a). While all of the newly-identi®ed genes respond toIR in at least one cell line in addition to ML-1, twoof the cell lines, K562 and A549, did not show g-rayregulation of any of the newly-de®ned IR-responsive

genes, and only ATF3 was induced by any stress agenttested in these two lines (Figure 3b).

Interestingly, K562 was the least responsive of anyof the cell lines, and was the only line derived from apatient with chronic myelogenous leukemia. Of thenine genes screened, 5(SSAT, c-Myc Promoter bindingprotein (MBP-1, or PRDII-BF1) (Fan and Maniatis,1990), cellular inhibitor of apoptosis 1 (c-IAP1)

Figure 3 Regulation of transcripts identi®ed on the microarray measured in a panel of human cancer cell lines. Numbers shown arethe relative induction for each gene over levels in untreated controls 4 h after treatment with (a) 20 Gy ionizing radiation or (b) 100mg/ml methyl methanesulfonate (MMS) or 14 J/m2 ultraviolet (UV) radiation as measured by quantitative dot-blot hybridization(Hollander and Fornace, 1990). A zero indicates no detectable expression in either control or treated cells. The results are colorcoded: red for 4twofold induction, green for 4twofold reduction, and yellow for 5twofold change from untreated control

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(Rothe et al., 1995), RELB (Liou et al., 1994) andBCL3 (Zhang et al., 1994)) were primarily induced byIR in the 12 cell lines examined. Fos-related antigen-1(FRA-1) (Cohen and Curran, 1988), RCH1, andprohormone convertase1 (PC1) or neuroendocrineconvertase1 (NEC1) (Seidah et al., 1991), all showedregulation by the base-damaging agents MMS or UVradiation in some cell lines (Figure 3b). The most wideranging response was seen for activating transcriptionfactor 3 (AFT3) (Liang et al., 1996), which wasinduced by both MMS and UV radiation in all celllines tested. With such a strong and pervasiveresponse, ATF3 is likely to play an important role

in generalized genotoxic stress responses. The complexpatterns seen in Figure 3 emphasize the importance ofcellular context in genotoxic stress responses andhighlight the need for high throughput techniquesfor the analysis of these responses, since even withonly 12 targets, each stress and cell line had a uniquepattern of induction.

The induction patterns in Figure 3 may hold cluesto regulation of the induction of some of these genes.For instance, the e�ect of the p53 status of a cell onits ability to induce CIP1/WAF1 (El-Deiry et al.,1993; Zhan et al., 1995) and MDM2 (Chen et al.,1994b; Perry et al., 1993) in response to IR has beenwell documented. This e�ect is re¯ected in the weak toabsent IR-induction of these two transcripts in p53mutant cell lines in this panel compared to the clearIR-induction in all p53 wild type lines examined.Although not so widely induced, FRA-1 showed asimilar pattern in that it was not induced by IR inany of the p53 mutant lines in this panel. In addition,the ®vefold IR-induction of ATF3 in RKO wasattenuated in the RKO/E6 cell line, although someIR-induction was seen among the other p53 mutantcell lines. These results raised the possibility that theIR-induction of FRA-1 and ATF3 may involve a p53regulatory component. Figure 4a shows the IR-induction of ATF3 in the p53 wild-type humancarcinoma cell line RKO and in RKO/E6, in whichp53 function has been abrogated by an E6 expressionvector. Both these lines have been well characterizedin our laboratory (Smith et al., 1995; Zhan et al.,1993, 1994a), and RKO/E6 has been shown to lackappreciable functional p53. The disruption by E6 ofATF3 induction by IR supports a role for p53 in itsinduction. To further test the extent of dependence ofATF3 IR-induction on p53 status, we examined the invivo induction in wild-type and p537/7 (knockout)mice (Donehower et al., 1992) using 5Gy whole-bodyg-irradiation. While ATF3 was well induced by 2 hafter irradiation in the thymus of wild-type mice, therewas no signi®cant induction in the p537/7 mouse(Figure 4a). ATF3 levels remained elevated at 4 and8 h after irradiation in the thymus of wild-type mice,without any induction in the p537/7. A similartrend was seen in the liver, where ATF3 was inducedabout fourfold in the wild-type mouse, and notinduced in the p53 knockout (data not shown).

ATF3 is a member of the activating transcriptionfactor/cAMP response element binding protein (ATF/CREB) family which homodimerizes to represstranscription from promoters with ATF sites. Analternatively spliced form of the ATF3 transcript,which lacks DNA binding activity, is also expressedin cells, but this form promotes transcription (Chen etal., 1994a). Based on the sizes of ATF3 transcriptshybridizing on the Northern blot, the smalleralternatively spliced form was the major transcriptexpressed in untreated ML-1 cells, whereas the IR-induced transcript was predominantly of the full lengthform (Figure 2). Full length ATF3 can also form aheterodimer with the stress-protein Gadd153, prevent-ing its usual transcriptional repression (Chen et al.,1996). ATF3/Jun heterodimers, on the other hand,activate transcription in transient transfection assays(Hsu et al., 1992). Adenovirus E1A (AdE1A) expres-sion also induces ATF3, and leads to the formation of

Figure 4 (a) Induction over basal levels of ATF3 4 h after 20 GyIR in p53 wild-type RKO cells (light bar), or in RKO/E6 (darkbar), in which p53 function has been abrogated by an E6expression vector, and 2 h after IR in the thymus of p53 wild-typemouse (light bar), or p537/7 (dark bar). The results shown forthe RKO cell lines are the average of four independentexperiments, and error bars are standard errors of the means.The dotted line marks the basal level corresponding to a relativeinduction of 1.0. (b) Induction over basal levels of FRA-1 in p53wild-type MCF7 cells (light bar), or in MCF7/E6 (dark bar) inwhich p53 function has been abrogated by an E6 expressionvector, and in the thymus of p53 wild-type mouse (light bar), orp537/7 mouse (dark bar). The results for the MCF7 cell linesare the average of three independent experiments

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cJun/ATF3 heterodimers, possibly contributing tooncogenic transformation by AdE1A (Hagmeyer etal., 1996). ATF3 has previously been shown to beinduced by serum stimulation, 12-O-tetradecanoylphor-bol-13-acetate (Chen et al., 1994a), and by physiologi-cal stresses such as wounding, CC14 and alcoholintoxication, ischemia/reperfusion and brain seizure(Chen et al., 1996). The induction of ATF3 by theDNA-damaging agents MMS and UV radiation in all12 cell lines tested in this study extends the range ofstress responses in which ATF3 is involved. Its wide-ranging responsiveness is reminiscent of CIP1/WAF1and GADD45 which are stress inducible by both p53-dependent and independent mechanisms (Holbrook etal., 1996; Zhan et al., 1995), and highlights thecomplexity of ATF3 regulation.

Although FRA-1 was not IR-inducible in RKO cells,a similar comparison was possible using the MCF7 andMCF7/E6 cell lines. The human breast carcinoma cellline MCF7 has wild-type p53, while MCF7/E6 hasbeen demonstrated to lack appreciable wild-type p53function (Smith et al., 1995; Zhan et al., 1993, 1994a),and both cell lines have been well characterized in ourlaboratory. The reduced IR-induction of FRA-1 inMCF7/E6 compared to MCF7 again supports a rolefor p53 in the IR-induction of this gene (Figure 4b).Furthermore, whole-body g-irradiation of wild-typemice resulted in FRA-1 induction in the thymus, butno induction occurred in the thymus of the p537/7(Figure 4b). FRA-1 is an immediate early gene inducedby serum stimulation, the product of which sharesseveral regions of amino acid homology with Fos(Cohen and Curran, 1988). It has also been reported tobe down-regulated by UVB and upregulated by UVA(Ariizumi et al., 1996). Because of its homology to Fos,and the involvement of p53 in its IR-induction, FRA-1may represent a link between p53 and AP1 functionand the MAPK pathway.

MBP-1 represented another potentially p53 regu-lated gene, showing a pattern similar to that seen forBAX or BCL-X, in that it was induced only in p53-wild-type cell lines of lymphoid or myeloid lineage. Weexamined the induction of the murine homolog ofMBP-1 in the tissues of wild-type and p537/7 mice,and found marginal to absent expression in liver andthymus, but strong expression in spleen. Treatmentwith ionizing radiation resulted in a twofold inductionof this gene in the spleens of both p53 wild-type andp537/7 mice, suggesting that this gene does notrequire p53 function for its induction, but that itsexpression and induction are both limited to a subsetof cell types. This would be consistent with a role forMBP-1 in tissue speci®c p53-independent stressresponses.

The high variability of transcriptional responsesfound in di�erent cell lines (Figure 3) emphasizes thata single cell line or cell type cannot provide a generalmodel for cellular responses to genotoxic stress. Thefurther ®nding that only two of nine of these genesexamined in this cell line panel showed a recognizablep53 component to their regulation belies the recentfocus of stress-gene studies primarily on p53-regulatedgenes. For instance, another study, submitted shortlyafter this one, detected IR-regulation of multipletranscripts in human ®broblasts engineered to expresshigh levels of cloned p53, but induction of nearly all of

these genes was p53-dependent (Komarova et al.,1998). Interestingly, when this study was extended totissues of mice irradiated in vivo, the majority ofregulated genes found were responsive in a tissue-speci®c manner, again underlining the importance ofcellular context to stress-gene response. Other genediscovery approaches (Madden et al., 1997; Polyak etal., 1997) have also focused on p53-dependent geneinduction. Exclusive focus on such approaches usingengineered cell lines would likely overlook many IR-response genes, such as the majority of those describedhere in myeloid cells. In light of the loss of functionalp53 in the majority of tumors, the non-p53-dependentstress response genes may be an important considera-tion in cancer treatment.

Cellular context is clearly critical in determiningwhich genes can be induced, and stress responsesprobably involve multiple signaling pathways (Hol-brook et al., 1996; Zhan et al., 1996). It is likely thatthe patterns of transcriptional responses beginning toemerge in Figure 3 are indicative of as yetundiscovered cell-type-speci®c regulatory factors orthe interruption of signal transduction pathways insome cancer cell lines. Signaling pathways involvingp53, NFkB, the MAP kinases, and others have roles inboth protective and apoptotic responses after genotoxicstress (Holbrook et al., 1996; Smith and Fornace,1996b), and many of the genes responding on thismicroarray interact with members of these pathways.For instance, RELB, a modi®er of NFkB subunitbinding a�nity was induced in 10/12 cell lines studied,but only by IR. As NFkB activation is a fairlyuniversal response to genotoxic stress, transcriptionalregulation of this subunit may be one mechanism bywhich this activity is channeled into a more speci®cdownstream response. By changing the balance ofspeci®c NFkB heterodimers in the nucleus, an increasein RELB changes the a�nity of NFkB for promoterbinding, resulting in di�erential regulation of someNFkB targets (Ivanov et al., 1995; Liou et al., 1994). Ifan increase in RELB mRNA levels does occur only inresponse to an IR-speci®c signal or damage, this couldrepresent an important discriminator of stress-response.

In light of the complexity of cellular transcriptionalstress response highlighted in this study, quantitativefunctional genomics approaches, such as cDNAmicroarray hybridization, will be needed to unravelthe inter-relationships of the molecular responsepathways involved. Although radioactive-probe hybri-dization to nylon ®lter arrays provides a useful methodto screen for potential genes of interest which di�er inexpression levels between two samples, di�erentialscreening has its own limitations (Fargnoli et al.,1990); some of which are avoided by the use of probeslabeled with di�erent ¯uorochromes co-hybridized tothe same microarray. Other methods for identi®cationof di�erentially expressed mRNAs, such as di�erentialdisplay, subtractive library hybridization and serialanalysis of gene expression (SAGE) (Velculescu et al.,1995), can be biased toward detection of highly-expressed and/or strongly-induced transcripts. Withthe microarray approach reported here, quantitativeresults over a wide dynamic range were obtained formany genes. The application and further re®nement ofquantitative ¯uorescent cDNA microarray hybridiza-

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tion have the potential to advance our understandingof the ®elds of stress gene response and radiationbiology, and to extend this technology beyond simplepair-wise comparisons to applications such as tumortyping, pharmacological screening, biomonitoring, andrapid carcinogen screening.

AcknowledgementsWe would like to thank Stephen B Leighton, ThomasPohida and Paul D Smith for their contributions toinstrumentation design, Yuan Jiang and Gerald C Goodenfor their contributions to the microarray studies, andKhanh T Do for technical assistance. This work wassupported in part by DOE grant ER62683.

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