JOURNAL OF VIROLOGY, May 2010, p. 5131–5139 Vol. 84, No. 100022-538X/10/$12.00 doi:10.1128/JVI.01965-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Human Papillomavirus Type 16 E6/E7 Upregulation of NucleophosminIs Important for Proliferation and Inhibition of Differentiation�

Rachel McCloskey,1 Craig Menges,1† Alan Friedman,2 Daksha Patel,1 and Dennis J. McCance1*Centre for Cancer Research and Cell Biology, Queen’s University, Belfast BT9 7BL, United Kingdom,1 and Proteomics Center,

University of Rochester, 575 Elmwood Avenue, Rochester, New York 146422

Received 16 September 2009/Accepted 23 February 2010

The E6 and E7 oncoproteins of high-risk human papillomaviruses (HPVs) are together sufficient to causecellular transformation. Nucleophosmin (NPM) was identified as a protein with increased levels in two-dimensional (2-D) gel analysis of human foreskin keratinocytes (HFKs) expressing E7 following methylcellu-lose-induced differentiation. Analysis of NPM expression in E7-expressing cells and E6- and E7-expressingcells in culture and in organotypic rafts confirmed the increased levels observed in 2-D gel analysis. Theelevated expression of NPM was determined to be posttranscriptional and was attributed to increased v-aktmurine thymoma viral oncogene (AKT) activity in the E6- and E7-expressing cells. Depletion of NPM causeda reduction in the replicative capacity of E7- and E6/E7-expressing HFKs and an increase in markers ofdifferentiation. Also, the p53 and pRb tumor suppressor levels are increased with the knockdown of NPM inE6/E7-expressing cells, and, interestingly, p14ARF is relocalized from the nucleolus to the nucleoplasm andcytoplasm in these cells. The results show for the first time that NPM is required for the proliferation andinhibition of differentiation observed in HPV E6- and E7-expressing primary cells.

The E6 and E7 oncoproteins of human papillomavirus type16 (HPV-16) have been shown to cause immortalization ofprimary human keratinocytes and are expressed in malignantcancers caused by HPV-16 infection (27, 28). E6 is best knownfor its ability to bind and degrade the tumor suppressor p53,whereas E7 can inactivate the pRb family of tumor suppressors(2, 3, 26). E6 is one of the earliest genes expressed during HPVinfection and has been shown to bind sites at both the Cterminus and the DNA binding domain of p53. Degradation ismediated by the ubiquitin ligase E6-associated protein (E6-AP/UBC3A), leading to degradation of p53 via the 26S pro-teasome (14, 34). Another mechanism by which E6 inhibits p53activity is by binding to p300/CBP and inhibiting the coactiva-tion of p53-dependent gene transcription (30).

E7 can bind to and inactivate the pRb family of tumorsuppressors, Rb, p107, and p130 (5). These proteins play amajor role in regulating the cell cycle, transcriptional repres-sion, and tumor suppression (7, 11). E7 has the ability tooverride normal cell cycle activities by binding to the hypo-phosphorylated form of Rb, prematurely pushing cells into theS phase and resulting in disruption of differentiation. Recentdata have indicated the role of E7 in pRb-independent mech-anisms that target other cellular proteins and disrupt theirnormal function (1).

In an attempt to identify other significant targets of E7 wecarried out a two-dimensional (2-D) gel analysis of proteinsfrom E7-expressing primary human foreskin keratinocytes(HFKs) during methylcellulose-induced differentiation. Nu-

cleophosmin (NPM) was identified as a protein showing in-creased levels compared to the vector control cells. NPM is anucleolar phosphoprotein that is abundant in tumor and pro-liferating cells (9, 21). Although it is localized in the nucleoli,NPM has the ability to shuttle between the nucleus and cyto-plasm and can bind and chaperone proteins to alter theircellular localization (4). Regarded as a proto-oncogene, NPMis overexpressed in a range of cancers and is used as a markerfor colon, gastric, and ovarian cancers, with increased levels ofNPM correlating with tumor progression (8). It is also the mostfrequently mutated gene in acute myeloid leukemia (AML),with approximately 35% of patients showing an abnormality inthe gene (9). NPM functions through sustaining ribosome bio-genesis, inhibiting apoptosis and disrupting differentiation, andupregulation of NPM in cells leads to an increase in prolifer-ation (4). In this report, we provide the first evidence of a rolefor NPM in HPV-mediated proliferation and inhibition of dif-ferentiation. We show that NPM is upregulated by E7 at theprotein level through the ability of E7 to deregulate v-aktmurine thymoma viral oncogene (AKT) and that this upregu-lation is required for proliferation of cells and for the inhibi-tion of differentiation.

MATERIALS AND METHODS

Plasmids and siRNAs. The pBabe (puro), pBabe-E6stopE7 (E7), and pBabeE6/E7 retroviral constructs used were described previously (10). pSuper-retroconstructs expressing short-hairpin RNAs (shRNA) against no known annotatedgene (shScr) were cloned as previously described (31), as were the pSuper-retroconstructs expressing shRNAs targeting Rb and p53(15). The following se-quences were used for shRNAs targeting NPM: forward, 5�-CCA GTG GTCTTA AGG TTG AAG TGT GG-3�; reverse, 5�-TCC AGA TAT ACT TAAGAG TTT CAC ATC CTC CTC C-3�. Before transfection into �NYX-GPpackaging cells, all retroviral plasmid constructs were sequenced. Small interfer-ing RNAs (siRNAs) targeting AKT (SignalSilence 6211, 6510, and 6511) werepurchased from Cell Signaling. siRNAs targeting NPM (sense, UGA UGA AAAUGA GCA CCA G) and a Scrambled control (ACG GUA ACA GUC ACUGAG C) were designed and purchased from Darmacon.

* Corresponding author. Mailing address: Centre for Cancer Re-search and Cell Biology, Queen’s University, Belfast BT9 7BL, UnitedKingdom. Phone: 44 2890972184. Fax: 44 2890972776. E-mail: d.mccance@qub.ac.uk.

† Present address: Fox Chase Cancer Centre, Philadelphia, PA19111.

� Published ahead of print on 17 March 2010.

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Cell culture. Primary human foreskin keratinocytes (HFKs) were isolatedfrom neonatal foreskin and transduced with retrovirus produced using the�NYX-GP packaging line as previously described (10).

Differentiation of HFK cell lines and induction of differentiation. pBabe shScr,pBabe shRNA targeting NPM (shNPM), E6/E7 shScr, and E6/E7 shNPM HFKcell lines were induced to differentiate by using organotypic rafts as previouslydescribed (10). Raft cultures were harvested, fixed in 4% paraformaldehyde,embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E).Bromodeoxyuridine (BrDU) (20 �mol/liter) was added to the raft culture 12 hbefore harvest to label DNA-synthesizing cells. Cell lines were also induced todifferentiate by suspension in 1.6% methylcellulose (29).

Mass spectrometric sequencing of proteins extracted from silver-stained poly-acrylamide gels. Proteins were extracted from gel samples and digested intotryptic peptides as previously described (35). The tandem mass spectrometry(MS/MS) spectra were searched using SEQUEST7 and the Bioworks browser(both from Thermo Corporation, San Jose, CA) and the publicly availableEuropean Bioinformatics Institute nonredundant human.fasta sequence data-base (http://www.ebi.ac.uk/IPI/IPIhuman.html) to determine possible sequencecorrelations of known proteins.

Western blot analysis. Protein lysate concentrations were either 30 or 50 �gfor all blots, as described previously (29). In this study, the following primaryantibodies were used: mouse monoclonal anti-Rb and mouse monoclonal anti-p53 (BD PharMingen) (1:1.000); mouse monoclonal anti-B23 and rabbit poly-clonal anti-C23 (1:1,000) and mouse monoclonal antiactin (1:20,000) (Santa Cruz

Biotechnology); mouse monoclonal anti-p14ARF (Neomarkers) (1:500); and rab-bit polyclonal anti-K1 (Covance) (1:5,000). Secondary antibodies used in thisstudy were goat anti-mouse horseradish peroxidase (HRP) and goat anti-rabbitHRP (Santa Cruz Biotechnology) (1:2,000). Luminescence was detected by ei-ther Perkin-Elmer or Pierce enhanced chemiluminescence (ECL), and the signalwas detected using an Alpha Innotech FluorChem SP imaging system.

Real-time reverse transcription-PCR (RT-PCR) analysis. RNA was extractedwith a High Pure RNA isolation kit (Roche), according to the manufacturer’sinstructions. FastStart SYBR green Master (Roche) was used according to themanufacturer’s instructions to amplify PCR products, and fluorescence was mon-itored using a DNA engine Peltier thermal cycler (Bio-Rad) equipped with aChromo4 real-time PCR detection system (Bio-Rad). cDNA samples were di-luted 1:10 and quantified by amplification using a series of dilutions of controlcDNA. The following cycling conditions were used: initial denaturation at 95°Cfor 10 min, followed by 40 cycles of 95°C for 15 s, 58°C for 15 s, and 60°C for 60 s.Expression levels were assessed in triplicate and normalized to ribosomal largeprotein P0 (RPLP0) control levels. Graphs produced represent the combinedresults of three independent replicate experiments.

Metabolic labeling for NPM half-life determinations. pBabe- and E6/E7-expressing HFKs were pulse-labeled with 110 �Ci/ml of EasyTag EXPRESS35Sprotein-labeling mix–[35S]methionine-cysteine–2 mCi (74 MBq) stabilized aque-ous solution (catalog no. NEG772002MC; Perkin Elmer). After 3 h, the cellswere washed and labeled and were media chased with fresh unlabeled media.Cells were harvested at indicated time points thereafter, and immunoprecipita-

FIG. 1. Nucleophosmin levels are increased in E7- and E6/E7-expressing cells. HFKs were induced to differentiate by suspension in methyl-cellulose or organotypic rafts. A 2-D gel (a) and a magnified portion (b) showing an increase in NPM (circled in red and indicated with red arrows,respectively) in cells expressing E7 compared to pBabe control cells. (c and d) Western blots showing the expression of NPM in pBabe control cellsand E7-expressing cells during keratinocyte differentiation (c) or in cycling cells (d). (e) pBabe- and E7- and E6/E7-expressing organotypic raftsstained for NPM and DAPI, confirming the findings of the 2-D gel analysis. (Merged images show overlays of DAPI and NPM staining images).Scale bar, 100 �m. (f) NPM staining of normal and CIN3 cervical sections. NPM is upregulated in the CIN3 lesions compared to the paired normaltissue. Scale bar, 100 �m.

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tion (IP) and Western blotting were performed. Samples were subjected tosodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fol-lowed by staining in colloidal Coomassie blue (Invitrogen), and gels were driedunder vacuum conditions (Bio-Rad Geldryer). Dried gels were exposed to aPhosphoimager screen and read using a Fuji FLA-7000 analyzer. Bands werenormalized to protein levels in the immunoprecipitate.

Immunofluorescent staining. Paraffin-embedded organotypic raft sectionswere deparaffinized with 2 washes in xylene and rehydrated with 4 washes instep-down concentrations of ethanol. Antigen retrieval with citrate buffer (Dako)was performed for 20 min in a steamer. The sections were then washed threetimes in phosphate-buffered saline (PBS), and the primary antibody was applied.The following primary antibodies (diluted in 10% fetal bovine serum) were usedfor this study: rabbit polyclonal anti-keratin 1 (Covance) (1:4,000; no retrieval);mouse monoclonal anti-B23 and rabbit polyclonal anti-C23 (Santa Cruz Biotech-nology) (1:200); mouse monoclonal anti-BrDU (BD PharMingen) (for antigenretrieval; 1:200); rabbit polyclonal anti-Ki67 (LabVision) (1:200); mouse mono-clonal anti p14ARF (Neomarkers) (1:100); mouse monoclonal anti-p14ARF

(Sigma Aldrich) (1:100); and goat anti-mouse and anti-rabbit secondaries con-jugated to Alexafluor (488 nm or 594 nm) (Molecular Probes) (1:400). Cervicalintraepithelial neoplasia grade III (CIN3) lesions were described and processedas previously described (25). Images were taken using an Olympus BH-2 micro-scope and an Olympus D25 camera and Cell B software (Olympus). For raftsections, Ki67-positive cells were counted for a minimum of 15 fields of view(1,000 �M). For coverslips, a minimum of 15 fields of view and 400 cells werecounted for each slide and expressed as percentages of control numbers. Immu-nofluorescence images were captured using a Leica AF6000 inverted microscopeand Leica AF imaging software. Exposure times and deconvolution settings werekept constant within each experiment.

RESULTS

Nucleophosmin is increased at the protein level in E7- andE6/E7-expressing cells. In an attempt to elucidate data fornovel proteins that are upregulated in the presence of theHPV-16 E7 oncogene during differentiation, a proteomics ap-proach was taken. 2-D gel analysis of HFK lysates during 12 hof methylcellulose-induced differentiation revealed an increasein the levels of various proteins in cells expressing the E7oncogene compared to pBabe control cell results (Fig. 1a andb). Mass spectrometric analysis of the proteins revealed NPMto be one of the proteins whose level appears to be elevated indifferentiating E7 HFKs. To confirm these findings, HFKswere transduced with a retrovirus expressing either pBabe con-trol vector or HPV-16 E7 and cells were resuspended in meth-ylcellulose for 12 h to induce differentiation. Western blottingconfirmed an increase in NPM in E7-expressing differentiatingcells (Fig. 1c) and proliferating cells (Fig. 1d) compared topBabe control cells. Next, we wanted to determine whetherNPM levels are also elevated in E7 cells in a more biologicallyrelevant model system for keratinocyte differentiation, namely,an organotypic raft culture system. Sections of organotypicrafts generated from E7-expressing HFKs showed that NPMlevels were increased compared to the pBabe control raft levels(Fig. 1e). We also examined NPM levels in E6/E7-expressingcells, as these proteins are expressed in the context of HPV-16infection. Western blotting of proteins extracted from organo-typic rafts and immunofluorescence staining of sections fromorganotypic rafts revealed a substantial increase in NPM levelsin cells expressing E7 alone and in E6/E7-expressing cells com-pared to pBabe control cells (Fig. 1d and e). To determinewhether HPV-16-positive cervical lesions had increased NPMlevels, we used immunohistochemical staining of sections frommatched normal and cervical intraepithelial neoplasia gradeIII (CIN3) lesions. Investigation of the CIN3 lesions, all ofwhich were positive for HPV-16, revealed increased levels of

NPM compared to control epithelia within the same biopsyspecimen (Fig. 1f).

To determine whether the increase in NPM protein levelsobserved in E6/E7-expressing cells and rafts was due to in-creased transcription, RNA from three independent sets ofpBabe- and E6/E7-expressing HFKs was extracted for real-time PCR. NPM levels were normalized to an RPLP0 control,and values from the three individual experiments were aver-aged. Results showed that there was no significant increase inNPM RNA levels in E6/E7-expressing cells compared topBabe control cells, suggesting that the increase in NPM levelswas not due to increased transcription (Fig. 2a).

Increased NPM levels in E6/E7-expressing cells is due toincreased levels of active AKT. Having determined that theincrease in NPM observed in E6/E7-expressing cells was nottranscriptional, we next wanted to identify a possible mecha-nism to explain the increased protein levels of NPM in thesecells. Previous data from our laboratory show that E7 upregu-lates AKT activity through deregulation of pRb protein (25). Arecent report (20) has shown that AKT interacts with NPMand protects it from degradation. Therefore, either AKT wasdepleted or the activity was inhibited in pBabe- and E6/E7-expressing HFKs by small interfering RNAs (siRNA), an AKTinhibitor (AIV), and a PI3-kinase inhibitor (PI103). Reduced

FIG. 2. Increases in nucleophosmin levels in E6/E7-expressing cellsoccur at the protein level and may be due to increased AKT activity.(a) Real-time PCR was carried out on 3 different sets of pBabe- andE6/E7-expressing HFKs. Data shown in the graph represent averagesof the results of the 3 experiments and show that NPM mRNA levelsare not increased in E6/E7-expressing cells compared to pBabe controlcells (P � 0.13). (b) Depletion of AKT by siRNA (siAKT) decreasesNPM protein levels in pBabe- and E6/E7-expressing cells. (c and d)Reductions in AKT activity, but not total protein activity, by the use ofan AKT inhibitor (AIV) and a PI3-kinase inhibitor (PI103) reduceNPM protein levels.

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levels of AKT protein correlated with reduced levels of NPMin both pBabe- and E6/E7-expressing HFKs (Fig. 2b). In ad-dition, the treatment of cells with AKT or PI-3 inhibitorsresulted in a reduction of AKT activity, as measured by areduction in glycogen synthase kinase-3� phosphorylation(pGSK-3�), and a corresponding decrease in NPM proteinlevels (Fig. 2c and d). The half-life of NPM in control andE6/E7-expressing cells was measured in 35S-labeled cells andwas found to be 24 h in control cells, but over the same periodthe levels in E6/E7-expressing cells did not change (Fig. 3a). Inline with a role for AKT in NPM stability, when AKT wasdepleted and stability determined at 16 h after the 35S labeledamino acids were washed out, the level of NPM in depletedE6/E7-expressing cells dropped to control cell levels. However,no change in NPM localization was observed when AKT wasdepleted using siRNA (Fig. 3b). Indeed, NPM was localized to

the nucleolus in all cell types examined, including normal HFKsand pBabe- and E6/E7-expressing cells (data not shown).

Knockdown of NPM reduces proliferation and induces dif-ferentiation in E6/E7-expressing cells with upregulation of p53and pRb. Cells expressing E6/E7 have the ability to overridethe normal process of cell cycle control and differentiation. Todetermine the effect of knockdown of NPM on proliferationand differentiation, E6/E7-expressing HFKs were infected withretrovirus expressing scrambled short-hairpin RNAs (shRNA)expressing either a scrambled shRNA (shScr) or an shRNAtargeting NPM (shNPM) and stable cell lines were generated.Alternatively, NPM was depleted by RNA interference (RNAi)molecules (siScr and siNPM) and proliferation was investi-gated in short-term assays. The cell lines were tested for thelevel of proliferation as monolayers and in organotypic rafts bythe use of BrDU incorporation and Ki67 staining, respectively,

FIG. 3. Half-life of NPM in control and E6/E7-expressing keratinocytes. (a) The half-life of NPM is increased in E6/E7-expressing cellscompared to pBabe control cells but is reduced to pBabe levels in E6/E7-expressing cells when AKT is depleted by siRNA. Data in the graphrepresent averages of the results of 3 experiments from different time points; densitometry data represent percentages of the value for h 0 for thecontrol (pBabe). (b) NPM localization is unchanged by AKT depletion in pBabe- and E6/E7-expressing cells. Scrambled control and AKT-depletedcells were stained for NPM (green), nucleolar marker C23 (red), and DAPI (blue). Images show no change in NPM localization with AKTknockdown in pBabe- and E6/E7-expressing cells.

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while the RNAi-treated cells were studied only as monolayercultures. BrDU- and Ki67-positive cells were counted andquantified against DAPI (4�,6�diamidino-2-phenylindole)-pos-itive cells, in approximately 15 different fields of view. A 40%decrease in proliferation of cells in a monolayer (Fig. 4a and b)and a 50% reduction in proliferation of E6/E7-expressingHFKs in raft cultures compared to scrambled E6/E7-express-ing cell results (Fig. 4c and d) were observed. Similar reduc-tions in proliferation were also observed in pBabe cells whenNPM was depleted (Fig. 4e). Taken together, these resultssuggest that E6/E7-expressing cells with reduced NPM levelsare less proliferative than pBabe control cells.

To determine the effects of knockdown of NPM on differ-entiation, control or NPM-depleted HFKs were differentiatedby two different methods. First, the cells were grown to con-fluence and then treated with 1.5 mM CaCl2 to induce differ-entiation and harvested at various times thereafter. Alterna-tively, organotypic rafts were produced with the depleted cells.As expected, both Western blotting of proteins from CaCl2-treated cells and immunofluorescence analysis of sections fromorganotypic rafts showed low levels of K1 expression in theE6/E7-expressing scrambled control cells (Fig. 5a and b). In-terestingly, E6/E7-expressing cells with NPM knockdownshowed increased levels of K1 in both Western blot analysis of

CaCl2-treated cells and immunoflorescence staining of raftsections (Fig. 5a and b). These data show that when the ele-vated levels of NPM in E6/E7-expressing cells are reduced,there is a decrease in proliferation and cells start to differen-tiate. Therefore, NPM contributes to the inhibition of differ-entiation observed in E6/E7-expressing cells.

To address the mechanism of decreased proliferation andincreased K1 expression in E6/E7-expressing shNPM cells, weanalyzed the expression of p53 and pRb in these cells. InE6/E7-expressing HFKs, there were normally low levels ofboth p53 and pRb; this was confirmed by Western blotting(Fig. 5c). However, there was a modest but consistent increaseof both pRb (approximately 28% increase) and p53 (approxi-mately 24% increase) levels in NPM-depleted E6/E7-express-ing cells (Fig. 5c).

ARF is relocalized from the nucleolus to nucleoplasm inE6/E7-expressing cells with the knockdown of NPM. NPM is ashuttling protein that binds to and influences the localizationof a number of proteins, including p14ARF (ARF). Since ARFis regulated in part by Rb through E2F and since it stabilizesp53, we investigated the levels of ARF in E6/E7-expressingcells compared to control cells. ARF levels were significantlyhigher in E6E7 cells (Fig. 6a). Localization of ARF is impor-tant for biology, so both E6/E7-expressing cells with or without

FIG. 4. Knockdown of NPM decreases proliferation in E6/E7-expressing cells. NPM levels in cells were reduced by the presence of eithershort-hairpin molecules (shRNA) or small interfering siRNA. Organotypic rafts and cycling cells were stained with Ki67 and BrDU, positivelystaining cells were counted, and percentages compared to DAPI-positive cells were quantified. (a and b) Cycling cells showing a 40% reductionof BrDU incorporation in siNPM-expressing cells compared to scrambled control cells (siScr). Data represent results from 15 different fields ofview over the course of 3 different experiments. (c and d) Organotypic rafts of cells stably expressing shRNA with respect to NPM (shNPM) showeda 50% reduction of Ki67 expression in stable shE6/E7-expressing cells compared to scrambled control cells (shScr). Ki67-positive cells werecounted in 15 different fields of view. (e) Proliferation was also decreased by over 40% in pBabe-expressing cells with the knockdown of NPMsiNPM compared to scrambled control cells (siScr). Data in graphs represent averages of the results of 3 experiments.

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NPM depletion and control pBabe cells were analyzed forsubcellular localization of ARF. It should be noted that incontrol and E6/E7-expressing cells, NPM in HFKs was local-ized to the nucleolus and colocalized with the nucleolar markerC23 both in cycling (Fig. 6b) and during differentiation (datanot shown). In pBabe-expressing cells, ARF was localized tothe cytoplasm-nucleoplasm (Fig. 6b), whereas in E6/E7-ex-pressing shRNA control cells, ARF colocalized with NPM inthe nucleolus (Fig. 6b). In E6/E7-expressing cells with depletedNPM, ARF was relocalized to the nucleoplasm-cytoplasm(Fig. 6b). This suggests that in E6/E7-expressing shRNA con-trol cells, NPM sequesters ARF to the nucleolus and conse-quently reduces the stabilizing effect of ARF on p53 in thesecells. However, upon depletion of NPM in E6/E7-expressingcells, ARF relocalizes from the nucleolus to nucleoplasm,which may result in the stabilization of p53 observed in E6/E7-expressing HFKs with NPM knockdown (Fig. 5b). This relo-calization of ARF from the nucleolus to nucleoplasm was alsoobserved in E6/E7-expressing cells when AKT activity wasinhibited using the AKT inhibitor AIV (Fig. 7a), suggestingthat AKT activity and the ability to stabilize NPM are impor-tant for E6/E7 to relocalize ARF to the nucleolus.

To determine whether ARF localization to nucleoli in E6/E7-expressing cells was due to disruption of the p53 and pRbfunctions, we next generated HFK cells with knockdown of p53or of pRb or of both p53 and pRb. NPM levels increased only

when both p53 and pRb were depleted (Fig. 7b), while in cellswith only p53 or pRb depleted, NPM levels are similar tocontrol cell levels (data not shown). Interestingly, when bothpRb and p53 were depleted, ARF was localized to the nucleoli,as was the case in E6/E7-expressing cells (Fig. 7c). NPM re-mains in the nucleoli in these cells (data not shown). Theseresults suggest that abrogation of the activity of both p53 andpRb is required for the nucleolar localization of ARF observedin E6/E7-expressing cells.

DISCUSSION

The intricate balance between cell proliferation and differ-entiation is crucial for maintenance of homeostasis and normaldevelopment within the cell, and disruption of either of theseprocesses may result in oncogenesis. We and others have pre-viously reported on the ability of the E6 and E7 oncoproteinsto disrupt the normal process of differentiation of HFKs bytargeting key tumor suppressors such as p53 (28) and pRb (17,37), resulting in increased levels of cell survival proteins suchas AKT (25) and disruption of the cell cycle (24, 31). In thisstudy, we investigated the possible role of the nucleolar phos-phoprotein nucleophosmin (NPM) in the differentiation pro-cess of HFKs. Frequently overexpressed in tumors and highlyproliferating cells, NPM has previously been shown to reducethe susceptibility of cells to the onset of differentiation and

FIG. 5. Nucleophosmin knockdown induces K1 expression and increases p53 and pRb levels in E6/E7-expressing cells and organotypicrafts. HFKs were induced to differentiate either in media containing 1.5 mM calcium chloride or in organotypic rafts. (a) Western blotshowing expression of NPM, K1 (an early marker of differentiation), and actin in E6/E7-expressing keratinocytes during induced differen-tiation at 0, 12, 24, and 36 h after calcium treatment. K1 levels were increased in cells with reduced levels of NPM (E6/E7shNPM) comparedto the results seen with control cells (E6/E7shScr). (b) E6/E7-expressing organotypic rafts, sectioned and stained with H&E (upper panels)and K1 (lower panels), showing the induction of K1 with knockdown of NPM. (c) Western blots showing the expression of Rb and p53 inpBabe control and E6/E7-expressing cells with either a scrambled siRNA (siScr) or an siRNA directed to NPM (siNPM).

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apoptosis (13, 33). NPM has also been reported to play acrucial role in sustaining ribosome biogenesis in cancer cells(12) and is now regarded as important in the development ofvarious cancers (9, 13, 22), with the first reported NPM inhib-itor developed as an anticancer agent in 2008 (32). NPM wasidentified as one of the proteins that were also upregulated ina proteomic screening undertaken to characterize proteins al-tered in expression in differentiating cells expressing E7 andsubsequently shown to be upregulated when both E6 and E7are present. The function of E6 targeting p53 for degradation,combined with the ability of E7 to deregulate pRb and familymembers as well as AKT, has been extensively characterizedand is known to be required for inhibition of differentiationand promotion of proliferation. This report describes anotherpathway involving NPM that is utilized by cells with E6/E7 tomaintain proliferative capacity and to potentially circumventthe activity of p53.

Our data show that the upregulation of NPM observed inE6/E7 was not due to increased transcription. Recent work(20) has shown that AKT binds to NPM and prevents itsdegradation. Previous work had shown that E7 can upregulate

AKT through disruption of the pRb and family member func-tions (25). Therefore, it was logical to test whether this up-regulation of AKT was responsible in part for the elevatedlevels of NPM detected in E6/E7-expressing cells. Indeed,when AKT was depleted in E6/E7-expressing HFKs by siRNAor AKT activity was inhibited by either PI-3 kinase or AKTinhibitors, there was a marked decrease in NPM levels. Deple-tion of pRb alone did not cause an increase in NPM levels, andso it would appear that either another function of E7 is in-volved or the other pRb family members, p130 and p107, havea role to play.

The involvement of NPM in the increased proliferation anddisruption of differentiation in different types of cells has beenpreviously reported (9, 33). E6/E7-expressing HFKs can over-ride the normal process of differentiation, and in this presentreport we provide the first evidence that increased levels ofNPM in HFKs are important for inhibition of differentiationand proliferation. Stable or transitory knockdown of NPM byshRNA or siRNA molecules, respectively, in E6/E7-expressingHFKs led to a decrease in proliferation and an increase in thelevels of the differentiation marker K1. The data demonstrate

FIG. 6. ARF is relocalized from the nucleolus to nucleoplasm in E6/E7-expressing cells with the knockdown of NPM. (a) Western blot showingan increase in ARF levels in E6/E7-expressing cells. (b) pBabe- and E6/E7-expressing HFKs were treated with either scrambled control or NPMsiRNA. Cells were stained for ARF (green), nucleolar marker C23 (red), and DAPI (blue). ARF was found in the nucleoplasm and cytoplasm incontrol cells but in the nucleolus in E6/E7-expressing cells. However, ARF was relocalized in E6/E7-expressing cells from the nucleolus tonucleoplasm-cytoplasm when NPM was depleted using siRNAs E6 and E7 (E6/E7 siNPM), but no change was observed in the control cells (pBabesiNPM).

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a clear role for NPM in suppression of differentiation. NPM isa nucleolar phosphoprotein that shuttles between the nucleusand the cytoplasm and takes part in various cellular processes.It has several interacting partners, some of which can be se-questered to the nucleolus and rendered inactive (22). Onesuch target is the tumor suppressor ARF, which has multiplefunctions that are dependent on localization within the cell(16). ARF has been shown to bind to NPM through the samedomain that mediates Mdm2 binding and nucleolar localiza-tion and as a consequence inhibits the ability of ARF to mod-ulate p53- and pRb-associated growth arrest functions (18, 38).Also, NPM has been shown to directly impact levels of p53 (19)as well as interact with pRb (23, 36). Here we show thatknockdown of NPM in E6/E7-expressing HFKs results in in-creased levels of p53 and pRb. In support of our results, arecently developed inhibitor of NPM induced apoptosis, up-regulated p53, and caused reduced proliferation rates in anumber of cell types (32).

Interestingly, ARF localization is altered in HFKs, where,upon knockdown of NPM in E6/E7-expressing cells, ARF islocalized in the nucleoplasm and cytoplasm as opposed tobeing sequestered in the nucleolus. The underlying mechanismthat allows ARF to be sequestered to the nucleolus by NPM inE6/E7-expressing cells is the disruption of both p53 and pRb

functions, since knockdown of both of these proteins is re-quired for the localization of ARF to the nucleoplasm-cyto-plasm. However, the increased levels of NPM in E7 or E6/E7-expressing cells do not solely account for the ability of NPM tosequester ARF to the nucleolus, since E7-expressing cells withincreased NPM levels do not sequester ARF to the nucleolus.The fact that E7 does not sequester ARF to the nucleolus mayaccount for the observation of an increase in p53 levels inE7-expressing cells (6). Therefore, while E7 may upregulateNPM levels, E6 must carry out another activity that helpssequester ARF to the nucleolus. In conclusion, our resultsprovide the first evidence of a role for NPM in HPV-mediatedcarcinogenesis and highlight the potential therapeutic use ofan NPM inhibitor in cervical cancer.

ACKNOWLEDGMENTS

This work was supported by grants from the NIH (NIDCRDE15935) and Wellcome Trust (WT082840AIA).

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FIG. 7. ARF is relocalized in HFKs after inhibition of AKT or after depletion of both p53 and pRb. (a) ARF is relocalized from the nucleolusto nucleoplasm-cytoplasm in E6/E7-expressing cells treated with the AKT inhibitor AIV. (b) Western blot showing HFKs stably expressing shRNAfor both p53 and Rb (shp53/Rb); a reduction in the levels of both proteins and a corresponding increase in NPM protein levels was seen comparedto scrambled (shScr/shScr) control cells. (c) ARF localized to nucleoli in control HFKs with depleted levels of both Rb and p53, and the localizationwas the same as that seen in E6/E7-expressing cells.

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