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Gene expression correlating with response to paclitaxelin ovarian carcinoma xenografts

Maria Rosa Bani,1 Maria Ines Nicoletti,1

Nawal W. Alkharouf,2 Carmen Ghilardi,1

David Petersen,2 Eugenio Erba,1

Edward A. Sausville,3 Edison T. Liu,4 andRaffaella Giavazzi1

1Mario Negri Institute for Pharmacological Research, Bergamoand Milan, Italy; 2Advanced Technology Center, National CancerInstitute, Gaithersburg, MD; 3Developmental TherapeuticsProgram, National Cancer Institute, Rockville, MD; and4Genome Institute of Singapore, National University of Singapore,Singapore

AbstractWe have investigated gene expression profiles of humanovarian carcinomas in vivo during TaxolR (paclitaxel)treatment and observed a difference in expression. Nudemice bearing 1A9 or 1A9PTX22 xenografts were given60 mg/kg of paclitaxel. Therapeutic efficacy was achievedfor 1A9, while 1A9PTX22 did not respond. Tumor tissuesharvested 4 and 24 h after treatment were evaluated bycDNA microarray against untreated tumors. Paclitaxelcaused the modulation of more genes in 1A9 than in1A9PTX22 tumors, in accordance to their therapeuticresponse. Most gene expression alterations were detected24 h after paclitaxel administration and affected genesinvolved in various biological functions including cell cycleregulation and cell proliferation (CDC2, CDKN1A, PLAB,and TOP2A), apoptosis (BNIP3 and PIG8), signal trans-duction and transcriptional regulation (ARF1, ATF2, FOS,GNA11, HDAC3, MADH2, SLUG, and SPRY4), fatty acidbiosynthesis and sterol metabolism (FDPS, IDI1, LIPA,and SC5D), and IFN-mediated signaling (G1P3, IFI16,IFI27, IFITM1, and ISG15). The modulation of two rep-resentative genes, CDKN1A and TOP2A, was validated byNorthern analyses on a panel of seven ovarian carcinomaxenograft models undergoing treatment with paclitaxel.We found that the changes in expression level of thesegenes was strictly associated with the responsiveness topaclitaxel. Our study shows the feasibility of obtaininggene expression profiles of xenografted tumor models as a

result of drug exposure. This in turn might provide insightsrelated to the drugs’ action in vivo that will anticipate theresponse to treatment manifested by tumors and could bethe basis for novel approaches to molecular pharmacody-namics. [Mol Cancer Ther. 2004;3(2):111–121]

IntroductionEvaluation of the response to chemotherapeutic regimens isoften empirical, mainly based on changes in tumor massrather than on molecular effects elicited by the drugs. Thetumor’s intrinsic biological characteristics together withmany other variables are most likely to underlie differencesin sensitivity. Thus, a given treatment may affect multiplepathways and elicit complex molecular responses, whichare presumably different than cancer response to therapy.Tubulin binding agents are an important class of clinicallyused antineoplastic compounds. Among them, TaxolR(paclitaxel) is a microtubule-stabilizing agent that blockscell division by interfering with the function of the mitoticspindle through inhibition of microtubule dynamics (1–3).Recently, a variety of cellular and molecular effects ofpaclitaxel have been described. These include induction ofcytokines, tumor suppressor genes, and activation of signaltransduction pathways (4). Paclitaxel is particularly impor-tant in the therapy of ovarian carcinomas, but the efficacy ishampered by the relapsing cancers resistant to chemother-apy (5). Proposed mechanisms relevant to paclitaxelresistance include overexpression of MDR1 (P-glycoproteindrug efflux pump; Ref. 6), point mutations (7), anddifferential expression of h-tubulin isotypes (8). In addi-tion, mitotic checkpoint control (9) and p53 status (10, 11)might also contribute to the sensitivity to paclitaxel (4).Despite such findings, there is little evidence that thesemechanisms influence the responsiveness of paclitaxel inclinical settings, where the development of resistance mayinvolve alterations of multiple genes, prior to or occurringunder the treatment.

In general, analyses have focused on the contribution ofindividual genes to drug response. Only recently, themolecular profiling of tumor cells exhibiting differentresponsiveness to anticancer drugs has been made possibleby microarray technologies (12–14). Molecular analyses ofthe response to treatment has been mainly explored in vitroand with the drug tested at high concentration. In vitrostudies do not take into account the host environment incontributing to a given response, which is of clearly greaterrelevance to clinical settings. Host metabolism certainlyinfluences pharmacokinetics, pharmacodynamics, molec-ular response, and, ultimately, drug sensitivity. The useof xenograft models allows the study of the in vivobehavior of human tumor and hence evaluate theirresponse to chemotherapy under the influence of hostfactors (15, 16).

Received 5/20/03; revised 10/29/03; accepted 11/26/03.

Grant support: Italian Association for Cancer Research; ProgettoStrategico Oncologia, Ministero dell’Istruzione, dell’Universita e dellaRicerca, Consiglio Nazionale delle Ricerche (Italy); and Italian Foundationfor Cancer Research fellowship (C. Ghilardi).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

Requests for Reprints: Raffaella Giavazzi, Mario Negri Institute forPharmacological Research, Via Gavazzeni 11, Bergamo 24125, Italy.Phone: 39-035-319888; Fax: 39-035-319331. E-mail: [email protected]

Molecular Cancer Therapeutics 111

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In the present study, we sought to examine the molecularevents elicited by paclitaxel in vivo in models of humanovarian carcinoma. By investigating the effects on geneexpression after paclitaxel was given to mice bearingxenografted tumors, we have found that the type andmagnitude of expression changes revealed by microarraycorrelated with the response to treatment. Results show themodulation of genes as a function of time of exposure to thedrug, and while several genes are affected in the responsivexenografts, considerably less changes are detectable in thenonresponsive xenografts. Gene expression alterationsoccurring shortly after paclitaxel administration (24 h)allowed us to distinguish the responding xenografts fromthe nonresponsive xenografts without knowing theirmechanism of paclitaxel resistance.

We believe that this information might propose a usefulapproach to response prediction in drug developmentstudies and assist in the design of novel strategies for thetreatment of cancer.

Materials andMethodsAnimalsFemale NCr-nu/nu mice were obtained from the Animal

Production Colony, National Cancer Institute-FrederickCancer Research and Development Center (Frederick,MD). The mice were 8–10 weeks old and had a meanbody weight of 23 g (SD = 2). Throughout this study, nudemice were housed in filtered-air laminar flow cabinets andmanipulated following aseptic procedures. Proceduresinvolving animals and their care were conducted inconformity with the institutional guidelines that are incompliance with national (Decreto Legge No. 116, GazzettaUfficiale, Suppl. 40, Feb. 18, 1992; Circolare No. 8, GazzettaUfficiale, July 1994) and international laws and policies(European Economic Community Council Directive 86/609,Official Journal Legislation 358.1, Dec. 12, 1987; Guide forthe Care and Use of Laboratory Animals, U.S. NationalResearch Council, 1996).

Ovarian CarcinomaModelsThe 1A9 and 1A9PTX22 cell lines were kindly provided

by Dr. Tito Fojo (NIH, Bethesda, MD). 1A9 was originallyderived from A2780 human ovarian carcinoma cells (17),and 1A9PTX22 was subsequently selected as a paclitaxel-resistant variant by exposure of 1A9 to the drug (7).IGROV1 cells originally established from a previouslyuntreated surgical specimen (18) were obtained from theNational Cancer Institute Tumor Repository (Frederick,MD). The cell lines were grown in vitro in RPMI1640 supplemented with 10% fetal bovine serum and5 mM L-glutamine. HOC18, HOC22, HOC94/2, andMNB-PTX1 xenografts were established from ovariancarcinoma patients and maintained in nude mice asdescribed previously (19). HOC18 and HOC22 were frompatients who had not received paclitaxel-based chemother-apy, whereas HOC94/2 and MNB-PTX1 were establishedfrom paclitaxel-refractory cancers (20, 21).

DrugTreatment ofTumor XenograftsTumor xenografts were obtained by injecting 10 � 106

cells in 200 Al suspension (1A9, 1A9PTX22, and IGROV1) orby transplanting tumor fragments (HOC18, HOC22,HOC94/2, and MNB-PTX1) into the flanks of nude mice.The growing tumor masses were measured with a Verniercaliper, and the estimates of tumor weights were calculatedby the formula: tumor weight = (length � width2) / 2.When tumor weight reached 150–300 mg, nude mice wererandomized to receive paclitaxel or vehicle or remaineduntreated (Fig. 1). Paclitaxel (kindly provided by Indena,Milan, Italy) was prepared in a mixture containing 50%Cremophor EL (Sigma-Aldrich, Steinheim, Germany) and50% ethanol and further diluted with 5% glucose in waterimmediately before administration. Paclitaxel was given byi.v. injection at the single dose of 60 mg/kg. This treatmentdid not affect the health status of the mice as evaluated bylack of body weight loss. Mice were used either for tumortissue collection and gene expression analysis or to assesstumor responsiveness to the treatment (Fig. 1).

Collection of Tumor Tissue for Gene ExpressionAnalysis

To study the molecular response to paclitaxel, wecollected samples before and 4 and 24 h after theadministration of paclitaxel or the corresponding vehicle(Fig. 1). For each of the xenograft models, the tumors wereharvested from 4 mice/group and bulk tissues were snapfrozen in liquid nitrogen immediately after collection andlater used for cDNA microarray analysis (only 1A9 and1A9PTX22) and for Northern analysis (all the xenograftmodels).

Expression Analysis by cDNAMicroarraysTotal RNA and Antisense RNA. Frozen tissue was

directly added to TRIzol reagent (Life Technologies, Inc.,Gaithersburg, MD) and homogenized with a tissue grinder.Total RNA was then isolated following the manufacturer’sinstructions and 3–4 Ag were amplified to obtain antisenseRNA (aRNA), essentially following the Eberwine protocol(22) with some modifications (23). First-strand cDNAsynthesis was obtained in a reverse transcription reactionprimed by oligo-(deoxythymidylate)24-T7 (5V-GGC CAGTGA ATT GTA ATA CGA CTC ACT ATA GGG AGGCGG-(T)24-3V), incubating the reaction at 42jC for 60 min inthe presence of SuperScript II. Double-stranded cDNA wasthen synthesized at 16jC for 2 h in the presence ofEscherichia coli DNA polymerase I, E. coli DNA ligase,and RNase H followed by 5 min with T4 DNA polymerase(all the enzymes were from Life Technologies). Double-stranded cDNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with ethanol inthe presence of 0.1 g of linear acrylamide. aRNA wasobtained by in vitro transcription with the T7 MEGAscriptkit (Ambion, Austin, TX) following the manufacturer’sinstructions. The reactions were allowed to take place for5 h at 37jC. To recover the amplified aRNA, an extractionwith phenol/chloroform/isoamyl alcohol (25:24:1) fol-lowed by a precipitation in ethanol was done. aRNAs werealiquot appropriately and stored at �80jC for future use.

In Vivo Gene Expression Changes Caused by Paclitaxel112



Both total RNA and aRNA concentrations were deter-mined by evaluating the absorbance at 260 nm and by gelelectrophoresis that also served to control for the quality ofthe samples.

Two-Color Fluorescent Hybridization. We fluorescentlabeled 3–4 lg of aRNA in a 40 ll reverse transcriptionreaction employing 400 units of SuperScript II in thepresence of 6 lg of random primer oligonucleotides[pd(N)6] and 4 pmol of cyanine 3-dUTP (Cy3) or cyanine5-dUTP (Cy5; Enzo Diagnostics, Farmingdale, NY). Reac-tion products were purified by repeated washes with10 mM Tris (pH 7.6)-1 mM EDTA in Microcon YM-30columns (Amicon/Millipore, Bedford, MA). The last washwas done by combining the appropriate experimentaltarget with the reference and concentrating the samplesdown to 14 ll in volume. Human CotI DNA (1 lg) andyeast tRNA (4 lg) were added to the purified and labeledsamples before heat denaturation. Hybridization to the

cDNAs microarrayed onto glass slide was carried out at65C for 14–16 h. Washes lasting 3–4 min each were donesequentially with 2� SSC and 0.1% SDS, 1� SSC, 0.5�SSC, and 0.05� SSC.

Cy5/Cy3 Fluorescence Ratio Evaluation. Fluorescenceimages and measurements of Cy3 and Cy5 were collectedby scanning the hybridized slides at 10 Am resolution on aGenePix 4000 microarray scanner (Axon Instruments,Union City, CA) at variable photomultiplier tube voltageto obtain maximal signal intensities with <1% probesaturation (Fig. 1). Resulting TIFF images were evaluatedusing the GenePix 3.0 software (Axon Instruments; Refs.24, 25) to quantify the extent of hybridization to any givencDNA spotted onto the slide (Cy5/Cy3 fluorescence ratio).Spots showing obvious defects were excluded from theanalysis. The generated raw data files were entered in aWeb-based relational database maintained by the Centerfor Information Technology, National Cancer Institute

Figure 1. Experimental design flow chart. Nude mice were implanted s.c. with the tumor of interest. Tumor-bearing mice were randomized at tumorvolume 150–300 mg to receive paclitaxel (60 mg/kg i.v.), vehicle, or no treatment. Groups of at least six mice were used for evaluating therapeuticresponse (see Fig. 2A). Additional groups of four mice were sacrificed before (0 h) and 4 and 24 h after treatment to collect tumor tissues for geneexpression analyses (see Fig. 2B). Examples of cDNA microarray hybridization for tumor xenografts 1A9 # 877 and 1A9PTX22 # 995 .




(Bethesda, MD). The expression ratios for the spots oneach array were normalized by subtracting the medianratio for the same array. Data were filtered to excludespots with a size of <25 Am and spots with an intensity ofless than twice the background or <250 units in both redand green channels.

For each of the 32 treated tumors, at least two (up to four)independent hybridizations were done, in which the targetand reference were labeled by exchanging the fluors. Thereference/control aRNA was from pooled equal amountsof total RNAs of tissue xenografts harvested from fouruntreated mice at the time of randomization.

Data Analysis. Comparison between groups was per-formed as follows. For each time point (4 and 24 h) andtreatment (paclitaxel and vehicle), xenografted tissues (1A9and 1A9PTX22) from four mice were evaluated against theuntreated tumors by means of the Hs-ATC-6.5k-4p humancDNA microarray manufactured at the National CancerInstitute Microarray Facility (http://nciarray.nci.nih.gov).On the Hs-ATC-6.5k-4p glass arrays, there are 6947 spottedcDNAs consisting of 5275 distinct clones, with 5097representing unique UniGene clusters (as for Build 160Homo sapiens) including 432 expressed sequence tags and4665 named genes. The additional 178 clones (mostlyunknown) are not present in UniGene.

The panel of genes that contribute to the expressionprofiles shown in Figs. 2B and 3 was selected based on

‘‘consistency of the observation.’’ For each treated mouse/tumor xenograft, only the cDNAs, the Cy5/Cy3 fluores-cence ratios of which reverted when target and referencewere labeled by exchanging the fluors, were consideredsignificant (to account for dye effects), and among them,only those with a ratio of 1.5 or greater were classified asoutlier cDNAs. To account for biological fluctuation amongtumor xenografts from different mice and to overcomevehicle-mediated effects, a cDNA/gene was then selectedout as relevant (Fig. 2B), only when the following twocriteria were simultaneously met: (a) it was an outlier in atleast three of the four paclitaxel-treated mice and (b) it wasnot an outlier in at least three of the four vehicle-treatedmice. The selected cDNAs/genes were then manuallyassigned to functional groups (Fig. 3).

As internal validation, self-hybridization and reversefluorochrome experiments were performed. Briefly, Hs-ATC-6.5k-4p human cDNA microarray from independentglass printing sets were hybridized to the same target byarbitrarily mixing Cy5 and Cy3 labeled aRNA (the aRNAused as the reference throughout the study). Results wereevaluated and analyzed to define the likelihood to find aCy5/Cy3 ratio for each individual spot altered in three offour slides. We found that none of the arrayed cDNAs werealtered when the cutoff for Cy5/Cy3 ratio was set at 1.5 orgreater. Thus, such a value was chosen as the limit toexclude variation due to experimental procedures.

Figure 2. Effect of paclitaxel on 1A9and 1APTX22 xenografts. 1A9 or1A9PTX22 were injected s.c. intonude mice. Tumor-bearing mice weretreated with paclitaxel (60 mg/kg i.v.).Details of the experimental design aredescribed in Fig. 1. A, antitumor effectagainst tumor xenografts. Points, me-dian RTW for six mice; arrows, day ofpaclitaxel administration. B, cDNAmicroarray analysis of xenografts taken4 and 24 h after paclitaxel administra-tion (4 mice/group). Colored images,graphical presentation of modulation ofgene expression. Color saturation isdirectly proportional to the magnitudeof the changes in the level of expres-sion evaluated against untreatedtumors: yellow, increased expression;light blue, decreased expression. Tu-mor xenografts are identified by thenumbers on top of each column,which are averaged Cy5/Cy3 valuesoriginating from at least two indepen-dent hybridizations for each treatedtumor.




Expression Analysis by Northern BlotTen to 12 Ag of total RNA from xenografted tissues were

electrophoresed through an agarose-formaldehyde dena-turing gel and transferred onto nylon membranes. Thefilters were hybridized overnight at 42jC with 32P-labeledcDNA dissolved in 50% formamide, 5% dextrane sulfate,5� saline-sodium phosphate-EDTA [0.75 M NaCl, 0.05 M

NaH2PO4, 5 mM EDTA (pH 7.4)], 1� Denhardt’s solution

(BSA, Ficoll, and polyvinylpyrrolidone, 0.2 mg/ml each),1% SDS, and 100 Ag/ml denatured salmon sperm. Filterswere then washed twice at 65jC with 0.2� SSC and 0.1%SDS. Probes for CDKN1A (p21/Waf1/Cip1/Sdi1) andtopoisomerase 2 a (TOP2A; 26) were kindly supplied byDr. M. Broggini (Mario Negri Institute for PharmacologicalResearch, Milan, Italy). To account for the amount of RNAbeing analyzed, the filters were hybridized overnightessentially following the protocol of Church and Gilbert(27) with a 32P-labeled oligonucleotide specific for 18SrRNA and washed thrice at 50jC (28). For each tumormodel, at least three tissues for each time point wereanalyzed (before and 4 and 24 h after paclitaxel delivery).The intensities of the bands on the autoradiographic filmwere evaluated by densitometry analysis using the Gel-ProAnalyzer software (Media Cybernetics, Silver Spring, MD).The ratio between the density of the mRNA of interest andthe corresponding rRNA was calculated, and it wasarbitrarily assumed that in untreated xenografts, this ratiocorresponded to 100% expression.

Cell Cycle StudiesTo study the cell cycle perturbation in response to

paclitaxel, we collected 1A9 xenografted tumor tissues

before and at different time intervals after the adminis-

tration of paclitaxel (4 and 24 h and 2, 4, and 6 days).

Immediately after collection, the tumors were dissociated

with 200 units/ml collagenase (Sigma Chemical Co., St.

Louis, MO) and the cell suspensions were fixed in 70%

ethanol and kept at 4jC until staining. DNA flow

cytometric analysis was performed as described previous-

ly (29). Briefly, the fixed cells were centrifuged, washed in

PBS, and stained with 2 ml of propidium iodide (PI)

solution containing 10 Ag/ml PI in PBS + 25 Al RNase

(10,000 units) overnight in the dark. The cell cycle phase

perturbations induced by paclitaxel were evaluated by

using a FACS Calibur instrument (Becton Dickinson,

Sunnyvale, CA). The fluorescence pulses of PI were

detected using a bandpass filter at 620 nm. Monopara-

metric DNA analysis was performed on at least 20,000

cells for each sample and analyzed with Cell Quest

software.

Tumor Response EvaluationMice were examined thrice a week. The end point of the

experiments occurred when tumors reached a weight off2 g. Tumor weights were normalized in the different

groups by obtaining the relative tumor weight (RTW)

calculated by the formula: RTW = W t / W0, where W t is the

tumor weight at any day of measurement and W0 is the

tumor weight at the start of treatment. The median RTW

(n = 6) for all groups was used to plot graphs evaluating the

efficacy of treatment. The %T/C (where T and C are

median RTW values for paclitaxel-treated and vehicle

controls, respectively) was calculated for all the mea-

surements and the lowest value was considered as opti-

mal growth inhibition in Table 1 (20). Net log cell kill

(NLCK) was calculated as [(T � C) � (duration of treat-

ment) � 0.301 / doubling time] and growth delay (GD)

Figure 3. Changes in gene expression caused by paclitaxel treatment.A, restricted to sensitive 1A9 xenografts; B, common to both 1A9 and1A9PTX22 xenografts; C, restricted to resistant 1A9PTX22 xenografts.Gray bars, increased expression level; black bars, decreased expressionlevel.




was calculated as [(T � C) / C � 100], where T and C aremedian times to reach 1 g, respectively, for treated(paclitaxel, n = 6) and controls (vehicle, n = 6). TR50 (tumorregression) indicates the percentage of mice with >50%reduction of tumor mass.

ResultsOverall MicroarrayAnalysis ofTumor XenograftsForty tumor tissue samples were collected and about 0.5

million measurements of cDNA expression were made. Theresults were reproducible in repeated hybridization experi-ments (data not shown), demonstrating the applicability ofsuch technology and procedures to tissues obtained ex vivofrom mice bearing xenografted tumors. Throughout thewhole study, analyses were conducted against referenceaRNA from xenograft tumors of untreated mice (Fig. 1). Foreach of the tumor xenografts collected after paclitaxel orvehicle administration, only the Cy5/Cy3 fluorescenceratios, the values of which reverted, were accounted for.The 1.5 fluorescence ratio was chosen as the cutoff value onthe basis of preliminary setup experiments, indicating thisas the limit to exclude variation due to experimentalprocedures (see ‘‘Materials and Methods’’ for details). Toovercome the biological variability among tumor tissuesfrom different mice, we evaluated four treated xenograftsfor each experimental group (Fig. 1). We have selected onlythose gene expression alterations simultaneously present inthree of four (75%) paclitaxel-treated mice but not affectedby the vehicle.

Changes inGene Expressionafter PaclitaxelAdminis-tration andTreatment Outcome in1A9 Xenografts

Mice bearing the 1A9 ovarian carcinoma xenograft weretreated with 60 mg/kg of paclitaxel given by single i.v.injection. The evaluation of tumor response showed thatpaclitaxel inhibited tumor growth in all the mice (T/C =12%, NCLK = 2.5, GD = 110%) with a >50% reduction of thetumor mass (TR50) in half of them (Fig. 2A; Table 1). This iswithin the expected range of response to paclitaxel on thepart of this model (30).

The evaluation of molecular response, by way ofcDNA microarray analysis of the 1A9 tumor xenograftscollected shortly after such a treatment, indicates thatpaclitaxel induced several changes. The level of mRNAexpression was altered in a variety of genes (78 cDNAs)and in a time-dependent fashion (Figs. 2B and 3). Fourhours after paclitaxel administration, 26 cDNAs wereaffected. Of those, about 30% remained clearly altered,most of them involved in signal transduction andtranscriptional regulation (Figs. 2B and 3). At longer timeafter paclitaxel administration, the number of changesincreased, most alterations (61 cDNAs) becoming evidentby 24 h (Figs. 2B and 3). At this time, changes mainlyaffected genes involved in proliferation/differentiation,apoptosis, cell metabolism, protein biosynthesis, andtrafficking (Fig. 3).

Modulation of genes involved in transcriptional

response affected both activators and repressors and was

already detectable 4 h after paclitaxel administration. The

zinc finger protein early growth response 3 (EGR3) was up-

regulated, but only transiently, as it returned to normal

expression level by 24 h. Instead, the expression of the zinc

finger protein SLUG decreased and remained low through-

out the 24 h. Similarly, the histone deacetylase HDAC3 , the

sprouty homologue SPRY4 , the G protein GNA11 , and

the LLR family member SSP29/APRIL were all induced at

4 h and remained overexpressed throughout the 24 h. In

addition, by 24 h, both c-fos and the c-fos interacting

upstream transcription factor USF2 , together with the

ADP-ribosylation factor ARF1, the transcriptional regulator

TBR1 and the activating transcription factor ATF2 , were all

down-regulated (Fig. 3).

Distinct genes involved in cell cycle regulation and cell

proliferation/differentiation were affected at either 4 or 24

h after paclitaxel delivery. By 4 h, there was an increase in

the expression of Nek1 [a protein kinase related to fungal

cell cycle regulator NIMA (never in mitosis A)] and adecrease in the expression level of the cell division control

CDC2-like protein kinases CLK-1 and CLK-2 and of the

dominant-negative helix-loop-helix proteins ID2 and ID3 .

Their modulation was transient, and as time progressed,

they all returned to normal expression level. Meanwhile, at

24 h, the protein kinases CDC2 (CDK1/p34cdc2) and

TOP2A were down-regulated and the cyclin-dependentkinase (cdk) inhibitor CDKN1A (p21/Waf1/Cip1/Sdi1), the

transforming growth factor-h (TGFh) superfamily member

PLAB , and the type IV protein phosphatase PRL1/PTP4A1

were up-regulated (Fig. 3).

Genes involved in apoptosis were affected 24 h afterpaclitaxel administration but not yet at 4 h. The expressionlevel of the negative controller of cell growth PIG8increased; conversely, the bcl2 family member BNIP3 wasreduced (Fig. 3).

The diminished expression of several IFN-induciblegenes such as G1P3 , ISG15 , IFI27 , IFITM1 , and IFI16 wasobserved 24 h after paclitaxel. At that time, numerousgenes involved in metabolism of fatty acids and sterol

Table 1. Therapeutic response of human ovarian carcinomamodels to paclitaxel

Xenografta Paclitaxel Responsivenessb

% T/C NLCK % GD TR50

1A9 12 2.5 110 50HOC22 9 2 520 100HOC18 22 1.2 90 60IGROV1 45 0.8 60 301A9PTX22 70 0.2 10 0HOC94/2 81 0.04 5 0MNB-PTX1 90 0.04 5 0

aTumor-bearing mice were randomized to receive 60 mg/kg of paclitaxel or vehicle(n = 6).bCriteria for therapeutic activity: %T /C , optimal growth inhibition < 10 = optimal;optimal growth inhibition < 25 = good, and optimal growth inhibition < 50 =moderate. NLCK < 1 is considered inactive. % GD, percentage of growth delay.TR50, percentage of mice with tumor mass reduction greater than 50%.




were also down-regulated. These included for examplethe dimethylallyltranstransferase/farnesyl diphosphatesynthase FDPS , the isopentenyl-diphosphate y isomeraseIDI1 , the cholesterol esterase lipase-A LIPA , and thesterol C5 desaturase SC5D. An exception was the lipo-protein lipase LPL , the expression of which decreased 4 hafter paclitaxel delivery but was back to normal by 24 h(Fig. 3). The expression of several genes involved inprotein biosynthesis and trafficking also decreased at 24 h,exceptions being the two ribosomal proteins RPS21 andRPS29 that were overexpressed (Fig. 3).

Changes in Gene Expression after Paclitaxel Ad-ministration and Treatment Outcome in 1A9PTX22Xenografts

Paclitaxel (60 mg/kg i.v.) given to 1A9PTX22-bearingmice did not achieve significant tumor response (T/C =70%, NCLK = 0.2, GD = 10%, TR50 = 0; Fig. 2A; Table 1).This confirms the nonresponse to paclitaxel expected forthis model (31).

Accordingly, the microarray analysis of the 1A9PTX22tumor xenografts collected shortly after paclitaxel adminis-tration showed that such a treatment caused only minimalperturbation in gene modulation. Changes in the expres-sion level occurred in a few genes at both 4 and 24 h (9 and7 cDNAs, respectively) after paclitaxel administration.Overall, only 40% (6 cDNAs) seemed selectively alteredin 1A9PTX22 xenografts (Fig. 3C), while about 60%(10 cDNAs) were affected also in 1A9 tumors (Fig. 3B).

Gene expression alterations induced by paclitaxelspecifically in 1A9PTX22 xenografts included the over-expression of the death domain CRADD adaptor and thedown-regulation of transcription factor TFAP4 and of TCL6occurring at 4 h. Overexpression of succinate dehydroge-nase SDHD , phosphatidylcoline transfer protein PCTP ,and SEC61G secretory protein was seen at 24 h (Fig. 3C).

Modulation affecting 1A9PTX22-resistant and 1A9-sensitive xenografts included the overexpression of thephosphogluconate dehydrogenase PGD and the down-regulation of ID2 and ID3 occurring at 4 h. In 1A9PTX22,the diminished expression of the IFN-inducible protein IFI27and the glucose transporter SCL2A3/GLUT3 was observed at4 h while it occurred at 24 h in 1A9 xenografts. The expressionof ARF1 and the major histocompatibility complex class IHLA-B decreased at 24 h for both xenograft models.Interestingly, PK428 serine/threonine protein kinase, theexpression of which decreased in 1A9PTX22-resistant tumors24 h after paclitaxel administration, was instead up-regulatedin 1A9-responsive tumors at both 4 and 24 h (Fig. 3B).

Gene Expression ChangesValidated by Northern BlotCDKN1A and TOP2A , the expression of which increased

and decreased, respectively, after paclitaxel treatment in1A9-responsive tumors (Fig. 4A), were chosen as repre-sentative genes to validate the microarray results. For thispurpose, 1A9 tissue xenografts (n = 12) were evaluated byNorthern blot analysis. As shown in Fig. 4B, the Northernanalysis of 1A9 xenografts confirmed that (a) 24 h afterpaclitaxel administration, CDKN1A expression increasedin three of four tumors whereas TOP2A decreased in all

four tumors, (b) these changes were not yet evident at 4 h,and (c) the magnitude of the modulation paralleled themicroarray results for the xenografts taken singularly(CDKN1A expression: 877 > 821 > 827 and TOP2A expres-sion 904 < 821 = 827 = 877; Figs. 4, A and B, and 5). Nochanges were detectable in vehicle-treated 1A9 xenografts(data not shown). These observations are in accordancewith a G2-M block induced in 1A9 tumor by the treatment.This was evident at 24 h after paclitaxel administration asshown by the DNA histograms in Fig. 4C.

Figure 4. CDKN1A and TOP2A expression and cell cycle perturbationfollowing paclitaxel administration. 1A9 or 1A9PTX22 were injected s.c.into nude mice. Tumor-bearing mice were randomized and treated withpaclitaxel (60 mg/kg i.v.). Details of the experimental design are describedin Fig. 1 and in Materials and Methods. A, CDKN1A and TOP2Aexpression by cDNA microarray analysis of 1A9 and 1A9PTX22xenografts taken 4 and 24 h after paclitaxel administration. For eachtreated tumor (identified by Mouse # ), the level of CDKN1A and TOP2Aexpression is presented in gray shaded boxes . Color saturation isproportional to the magnitude of the change in expression evaluated bycDNA microarray against untreated tumors (black, no difference). Forthose xenografts where change in expression is detectable, the Cy5/Cy3values calculated by averaging the Cy5/Cy3 ratios from 2 up to 4independent hybridizations are shown. B, CDKN1A and TOP2A expressionby Northern blot analysis of 1A9 xenografts. Nylon membranes with 10–12 Ag of total RNA (from xenografted tissues collected before or 4 and24 h after paclitaxel administration) were hybridized with 32P-labeledCDKN1A, TOP2A, and 18s rRNA probes. The autoradiographic signals areshown (for densitometric analysis, see Fig. 5).C, cell cycle analysis of 1A9xenografts. DNA flow cytometric analysis was performed for 1A9xenografted tumor tissues. Representative cell cycle phase distribution oftumors collected before and 4 and 24 h after paclitaxel administration.




In contrast to sensitive 1A9 tumors, microarray datashowed that in 1A9PTX22-resistant xenografts, neitherCDKN1A nor TOP2A expression was affected by paclitaxel(Fig. 4A). Concordantly, Northern blots of paclitaxel-treated 1A9PTX22 xenografts showed no changes in theexpression of TOP2A (Fig. 5). The expression level ofCDKN1A was below the detection capacity of the system inboth untreated and treated tissues.

Gene Modulation Is Confirmed in a Panel of OvarianCarcinoma Xenografts Treated with Paclitaxel

To extend our findings, we studied the effect of paclitaxelon CDKN1A and TOP2A modulation in a panel of ovariancarcinoma xenografts exhibiting different sensitivity to thetreatment. Paclitaxel (60 mg/kg i.v.) or vehicle was given totumor-bearing mice. Similar to 1A9, therapeutic efficacywas achieved for HOC18, HOC22, and IGROV1 xenografts(Table 1). Conversely, but in accordance with 1A9PTX22,the treatment with paclitaxel did not significantly affect thegrowth of HOC94/2 and MNB-PTX1 xenografts (Table 1).

Northern analyses revealed that by 24 h, the expressionof TOP2A decreased in HOC18, HOC22, and IGROV1(Fig. 5), all tumors highly responsive to the treatment withpaclitaxel (Table 1). Concomitantly, CDKN1A expressionincreased in HOC18 and IGROV1 (Fig. 5). CDKN1A was

below the level of detection in both untreated and treatedHOC22 tissues. No changes of CDKN1A and TOP2Aexpression were observed in HOC94/2 and MNB-PTX1tumors not responding to paclitaxel treatment (Fig. 5).

DiscussionThis study has analyzed at the transcriptional level humanovarian carcinoma xenografts undergoing treatment withpaclitaxel as a way of understanding the in vivo molecularconsequences of drug treatment. We have found thatpaclitaxel can affect the level of expression of a variety ofgenes and that such an effect occurred mostly 24 h after itsdelivery in those tumors responding to the treatment.These findings show that the therapeutic effectiveness ofpaclitaxel correlates with the dynamic modulation of geneexpression occurring shortly after its administration andalso suggest that evaluating the quantity of the changesand/or the change affecting specific gene types postche-motherapy in vivo might be a novel strategy to define/study pharmacodynamic end points.

To study drug responsiveness of cancer cells, moststudies have used cell lines in vitro often exposed to highdrug concentrations, not always achievable in the plasma ofpatients (4). In this study, 60 mg/kg of paclitaxel weregiven, a therapeutic relevant nontoxic dose for micebearing tumors (32), and by analyzing the gene expressionprofile of tumor xenografts from treated mice, this studytakes into account the pharmacokinetics and pharmacody-namics of the drug. After the administration of such a dose,paclitaxel rapidly reaches the tumors with a peak at 4 h andis still detectable 24 h later (data not shown). Different geneexpression kinetics were observed 4 and 24 h afterpaclitaxel administration that might either reflect the directeffects of the drug or result from downstream effectsmediated by earlier responding genes.

It is widely accepted that the cytotoxicity of tubulinbinding agents is due to their effect on the mitotic spindle,resulting in aberrant mitosis, aneuploidy, mitotic block,and apoptosis (1–3, 33). However, the underlying bio-chemical events leading to cell death after perturbation oftubulin are far from understood and a matter of currentinvestigation. Our study shows that the up-modulation anddown-modulation of genes involved in cell cycle regula-tion, cell proliferation, and apoptosis following paclitaxeladministration are consistent with an altered proliferationstate in the growth-inhibited tumors. Specifically, at 4 h, weobserved transient down-regulation of ID2 and ID3involved in coordination of proliferation and differentia-tion in mammalian cells. It has been shown that by bindingpRb pocket proteins, ID2 is able to abolish the S-phaseentry block (34). Conversely, the dual-specificity kinaseNek1, closely related to a cell cycle regulator that controlsinitiation of mitosis in Aspergillus nidulans (35), wastransiently up-regulated. In 1A9PTX22 xenografts, pacli-taxel caused only the transient down-regulation of ID2 andID3. It seems therefore that 1A9 but not 1A9PTX22 cellshave to deal with conflicting signals involved in controllingcell cycle progression and mitotic regulation. As a possible

Figure 5. Effect of paclitaxel on CDKN1A and TOP2A modulation in apanel of ovarian carcinoma xenografts. Experiments were performed asdescribed in Fig. 1. For each ovarian carcinoma model, at least threexenografted tumor tissues harvested before and 24 h after paclitaxel(60 mg/kg i.v.) were evaluated for CDKN1A, TOP2A, and 18S rRNAexpression by Northern blot analysis. The intensities of the bands on theautoradiographic film were evaluated by densitometry analysis. The ratiobetween the density of themRNAof interest and the corresponding rRNA foreach tissue was calculated. Columns, percentages of expression for eachtumor 24 h after paclitaxel administration in respect to the correspondinguntreated xenografts (closed bars, 100% expression). X axis labels:numbers under each column, number of the tumor xenograft; open bars,sensitive tumor models; striped bars, resistant tumor models; asterisks,below the limit of detection in both treated and untreated.




consequence, the attempt by 1A9 cells to traverse andcomplete the cell cycle is not going to succeed; while in theabsence of conflicting signals, cell growth will resume. Thisis supported by the perturbation of the necessary growthregulatory machinery that later occurred in 1A9 but not in1A9PTX22 tumors. Specifically, 24 h after paclitaxeladministration, different genes involved in cell growthregulation continued to be affected in 1A9 xenografts. Therelevance of such findings is supported by the cell cycleperturbation studies where a G2-M block in 1A9-sensitivexenograft occurs 24 h after paclitaxel administration. Theexpression of CDC2 and TOP2A decreased, whereas theexpression of CDKN1A increased. The TGFh superfamilymember PLAB (36), previously suggested to have a growthinhibitory influence, also increased. CDKN1A inhibits cdkactivity in such a way that the phosphorylation of G1 andG2 cdk critical substrates may be prevented (37). On theother end, CDC2, the primary mitotic kinase, is required toprogress through G2 and enter mitosis (38), where it alsoregulates the mitotic spindle and the movement ofchromosomes. The repression of TOP2A , which is de-scribed to be up-regulated during the G2 phase of the cellcycle, could certainly be a yet distinct mechanism ofmaintaining G2 arrest and prevent mitosis. Moreover, thephysical association of TOP2A/CDC2 contributes to theformation of precondensed chromosomes (39). Changes ingenes associated with apoptosis are also evident 24 h aftertreatment. The PIG8 (40) apoptosis inducer was up-regulated. Conversely, the expression of BNIP3 wasreduced. BNIP3 interacts with bcl2 and with E1B virus19-kDa protein and counteract the initiation of caspasecascade leading to apoptosis (41), and its down-regulationin response to apoptotic stimuli has been reported (42).Altogether, these alterations imply a complex interplayamong the different effects related to the action ofpaclitaxel.

Paclitaxel has been recently described to affect cellular

function not previously associated with microtubules, such

as cytokine release (43, 44) and transcription regulation

(45). However, these in vitro effects were seen with high

doses of paclitaxel. Our results were obtained in vivo , with

a clinically relevant dose of paclitaxel (32). In our study,

modulation of genes involved in the regulation of

transcription affected both transcriptional repression and

activation in 1A9-responsive tumors. Four hours after

paclitaxel treatment, HDAC3 was induced and remained

overexpressed throughout the 24 h. Meanwhile, at 24 h,

ATF2 , the motif of which responsible for stimulation of

transcription is localized within the histone acetyl transfer-

ase domain (46), was instead down-regulated. By impairing

access to chromatin and DNA through histone acetylation/

deacetylation changes, a mechanism to trigger a general

repression of transcription may be occurring in the cellular

response to paclitaxel. This might partly explain the general

decrease in levels of gene expression 24 h after paclitaxel

administration (Fig. 3), which is not seen in 1A9PTX22. On

the other hand, the decrease in expression of the transcrip-

tion corepressor SLUG (47) could suggest a more finely

tuned transcriptional activity. Transcription through TGFh-

responsive promoters and the c-fos-responsive element was

also affected by paclitaxel as indicated by the diminished

expression of MADH2/SMAD2 and both c-fos and its

interacting coactivator USF2 24 h after the administration

of paclitaxel. Beside its involvement in transcription,

HDAC3 contributes to the accumulation of cells in G2-M

phase (48), so its increased expression may also be im-

portant in perturbing cell cycle progression. Other impli-

cations of the unexpected link of paclitaxel action to

transcriptional activation through modulation of transcrip-

tional repressor mechanism might be an interaction

between taxane and histone deacetylase inhibitor drugs,

of which a number are entering early clinical trials (49, 50).

Finally, an action of paclitaxel with the cellular responseto stimuli mediated by transmembrane signaling systems issuggested by its effects on the small GTPase ARF1 and thetyrosine kinase receptor inhibitor SPRY4 , both part of theras-mediated signal transduction pathway (51, 52), and onthe guanine nucleotide binding protein GNA11 (53). It isalso worth noting the up-regulation of the transcriptioncoactivator EGR3 4 h after paclitaxel administration. AsEGR1 activates the PRL1 promoter (54), EGR3 might wellplay a role in the tyrosine phosphatase PRL1/PTP4A1increase seen at 24 h.

The link to modulation of ARF1 is also of interest in viewof the important role of ARF1 in maintaining the normalsecretory function of the cell through its regulation ofvesicle secretion and migration from the endoplasmicreticulum to the Golgi apparatus. Vesicles are associatedwith the microtubule network (55) and our data suggestthat perturbation of the microtubule network by paclitaxelmay induce a feedback/adaptive response by modulatingin part the formation of the vesicle coat processingmechanism. It is worth noting that CDC2 kinase candirectly phosphorylate dynein (minus-end-directed micro-tubule motor; 56) and its involvement in membraneorganelle movement during interphase has been suggested.Thus, CDC2 kinase down-regulation by paclitaxel mightalso be implicated in such phenomenon. Other signalingeffects are suggested by our data. The expression of severalIFN-inducible genes was reduced 24 h after paclitaxeltreatment of 1A9-responsive xenografts. Overexpression ofINF-responsive genes has been recently associated withresistance to paclitaxel in tumor cell lines (57). Thesefindings support the possible involvement of mediators ofINF signaling in responsiveness to paclitaxel.

Several genes involved in metabolism appear also to beaffected by paclitaxel. Interestingly, the expression of genesinvolved in sterol/cholesterol and fatty acid/glycerolipidbiosynthesis was diminished after treatment. While theeffect of a leaf and steam extract of Taxus baccata oncirculating cholesterol level has been reported (58), therelevance of this metabolic pathway to the mechanism ofaction of paclitaxel deserves further investigation.

The molecular effects elicited by paclitaxel documentedherein reflect genetic aspects of the response. Certainly,epigenetic mechanisms such as phosphorylation of proteins




linked to cell survival or activation of cell signalingpathways (4) are of importance to the drug’s effect butwould not manifest as changes in gene expression. Al-though the genes we analyzed are only a fraction of thosepresent in the whole genome, the investigation providesuseful information on the transcriptional response evokedby paclitaxel. As with any microarray-based strategy, re-sults are hypothesis generating and point to areas whereadditional experiments are required to assess the impor-tance of the changes.

Surprisingly, only a relatively small number of geneswere expressed differently in treated and untreatedxenografts. This might be due to the strict criteria imposedby our selection, which may have resulted in the exclusionof some genes. On the other hand, it should have avoidedexperimental artifacts and biological fluctuations, thusending with only a few genes relevant to the pharmaco-logical consequences of paclitaxel and not dependent onthe experimental model. The latter likelihood derivessupport from results on a panel of human ovariancarcinoma xenografts where, although only two geneswere examined, a strong correlation between the sensitivityto paclitaxel and the induction of CDKN1A and thedecreased expression of TOP2A emerged (Fig. 5). Thesefindings not only serve as one form of validation for themicroarray analysis results for 1A9 and 1APTX22 but alsoand most importantly extend the significance of theobservations to ovarian cancer, suggesting that geneexpression changes following drug delivery might havethe potential to distinguish responsive tumors from notresponsive ones. To this end, our findings argue against arole of p53 status in predicting the in vivo response topaclitaxel. p53 has been described as having different rolesin the sensitivity to paclitaxel depending on the experi-mental model used (10, 11). All the responsive ovariancarcinoma xenografts used here express wild-type p53(26, 59, 60) and paclitaxel treatment elicited a clear up-regulation of p53-responsive genes (CDKN1A and PIG8).Conversely, such an up-regulation was not seen inHOC94/2 and MNB-PTX1 xenografts, both using wild-type p53 but resistant to paclitaxel, or in the resistant1A9PTX22 expressing mutated p53 (Refs. 26, 60 and datanot shown).

It is noteworthy that our observations suggest that thedifferences in responsiveness as well as the effects on geneexpression cannot be easily attributed to the MDRphenotype, to impaired paclitaxel binding to h-tubulin, orto altered distribution of the h-tubulin isotypes expression.The whole panel of ovarian carcinoma xenograft modelsused here is negative for the MDR phenotype mediated bythe 170-kDa P-glycoprotein drug efflux pump overexpres-sion (7, 21, 26, 59). A point mutation in h-tubulin isotypeM40 has been described for 1A9PTX22 cells (7) and mightbe relevant to determine its resistance to paclitaxel. On theother hand, HOC94/2- and MNB-PTX1-resistant xenograftsexpress wild-type h-tubulin (21). In addition, h-tubulinisotypes expression distribution is similar in all the modelsused herein (7, 21).

To evaluate the patterns of gene expression in ovariancancer, we used in vivo models derived from ovariancarcinoma patients sensitive and resistant to paclitaxel.Some disadvantages of using an in vivo animal model mustbe considered. Drug metabolism and distribution in themice may be different from that of humans. However, theidentical genetic background of the mice, all bearingthe same tumor and receiving the same drug dose, ensuresthe reproducibility of the molecular pharmacology studies.In addition, the possibility of comparing tumor tissuesbefore and after treatment allowed us to study molecularmodifications along with the treatment.

In conclusion, this study shows the usefulness of mousexenograft human tumor models to study the molecularbasis for altered gene expression in response to a certaintreatment (in this case, paclitaxel). Our results measure themolecular consequences of the therapy rather than theinitial molecular differences in sensitive or resistant tumors(61). Molecular changes are potentially more selective andare detectable much sooner compared with the empiricallong-term evaluation of tumor growth inhibition. There-fore, in addition to informing about potential approaches tomodulating sensitivity to paclitaxel, they might serve asefficient pharmacodynamic molecular end points forevaluating drug activity shortly after drug administration.The relationships between antitumoral paclitaxel activityand gene expression levels are only correlative at this stage,but they generate hypotheses that are worth investigating.

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2004;3:111-121. Mol Cancer Ther Maria Rosa Bani, Maria Ines Nicoletti, Nawal W. Alkharouf, et al. ovarian carcinoma xenograftsGene expression correlating with response to paclitaxel in

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