[CANCER RESEARCH 61, 2356–2360, March 15, 2001]

Advances in Brief

Human Papillomavirus Type 16 E7 Oncoprotein-induced Abnormal CentrosomeSynthesis Is an Early Event in the Evolving Malignant Phenotype1

Stefan Duensing, Anette Duensing, Christopher P. Crum, and Karl Munger2

Department of Pathology and Harvard Center for Cancer Biology, Harvard Medical School, Boston, Massachusetts 02115 [S. D., K. M.], and Department of Pathology, SolidTumor Cytogenetics [A. D.] and Division of Women’s and Perinatal Pathology, Department of Pathology [C. P. C.], Brigham and Women’s Hospital, Harvard Medical School,Boston, Massachusetts 02115

Abstract

Genomic instability is a hallmark of malignant growth that frequentlyinvolves mitotic defects associated with centrosome abnormalities. How-ever, the question of whether abnormal centrosomes cause genomic insta-bility or develop secondary to other changes has not been conclusivelyresolved. Here we show that human papillomavirus (HPV)-16 E7 caninduce abnormal centrosome synthesis before the development of exten-sive nuclear abnormalities. In contrast, expression of HPV-16 E6 is asso-ciated with marked nuclear atypia and concomitant accumulation ofcentrosomes. Our results demonstrate that HPV-16 E7-induced centro-some abnormalities represent an early event during neoplastic progres-sion potentially driving genomic destabilization.

Introduction

The development of cervical cancer is tightly associated withinfection by high-risk HPV3 types such as HPV-16 or HPV-18,whereas low-risk HPV types like HPV-6 are associated with benigngenital warts. High-risk HPVs encode two oncoproteins, E6 and E7,which subvert crucial cellular regulatory nodes to reactivate andmaintain DNA synthesis in the host cell. Whereas high-risk HPV E6mediates the accelerated proteosomal degradation of the p53 tumorsuppressor protein, E7 binds to and destabilizes the retinoblastomatumor suppressor (pRB) and interferes with the cyclin-dependentkinase inhibitor p21Cip1 (1, 2).

Progression from normal to malignant cervical epithelium duringhigh-risk HPV-associated carcinogenesis is characterized by the de-velopment of genomic instability as manifested by abnormal chromo-some numbers,i.e., aneuploidy (3). The high-risk HPV E6 and E7oncoproteins can independently induce genomic instability in normalhuman cells (4–6). As in many cancers (7), genomic instability inhigh-risk HPV-induced lesions is associated with centrosome abnor-malities (8). The centrosome is the major microtubule organizingcenter in interphase and mitotic cells and assures symmetry andbipolarity of the cell division process by duplicating precisely oncebefore a cell division (9). Centrosome abnormalities have been de-tected in a wide variety of tumors; however, the mechanistic signifi-cance of centrosome abnormalities for the induction of genomicinstability has been debated. On one hand, it has been proposed thatabnormal centrosome numbers may directly cause mitotic defects that

lead to genomic instability. According to this model, centrosome abnor-malities emerge early during neoplastic progression in cells that do notyet show other manifestations of genomic instability. Alternatively, cen-trosome abnormalities may simply reflect genomic instability and accu-mulate in parallel with other cellular abnormalities. These two modelshave different implications for the diagnostic and prognostic value ofcentrosome abnormalities in human tumors. We have shown previously(8) that high-risk HPV E6/E7 oncoproteins cooperate to induce centro-some abnormalities and mitotic defects. Here we address the questionwhether E6- and E7-induced centrosome abnormalities drive the processof genomic instability or merely reflect the accumulation of cellularalterations. Expression of HPV-16 E7 is associated with an abnormalsynthesis of centrioles and induces abnormal centrosome numbers earlyduring neoplastic progression in primary human epithelial cells that donot display extensive nuclear abnormalities. In contrast, in HPV-16E6-expressing cells, centrosome abnormalities accumulate in cells withmarked nuclear atypia. These results support the model that HPV-16 E7causes mitotic abnormalities through dysregulation of centrosome home-ostasis early during malignant progression and therefore represents apotential driving force for genomic destabilization.

Materials and Methods

Cell Culture and Retroviral Infections. NHKs from neonatal foreskinswere isolated and cultured as described previously (2). For retroviral infectionof NHKs, recombinant retrovirus LXSN or LXSN-based retroviral constructsexpressing HPV-6 E6 or E7 or HPV-16 E6 or E7 were used (10). Forcoinfection of HPV-16 E6-expressing NHKs with HPV-16 E7, a high-titerpBABE E7 retroviral vector was produced as described previously (11).

The human U2OS osteosarcoma cell line was obtained from American TypeCulture Collection and maintained as described previously (8). Cells werestably transfected with a centrin-GFP plasmid (Ref. 12; kindly provided by M.Bornens; Institute Curie, Paris, France), followed by retroviral infection withpBABE E7 or empty vector used as control, and selected with puromycin.

Immunological and Cell Staining Methods. Cell lysates were made andanalyzed for expression of viral oncoproteins as described previously (2).Antibodies specific for p53 (Ab-6; Calbiochem, San Diego, CA) and HPV-16E7 (ED17; Santa Cruz Biotechnology, Santa Cruz, CA) were used.

Analysis of centrosomes in cultured cells was performed as describedpreviously usingg-tubulin-specific antibodies (Sigma, St. Louis, MO). Foranalysis of proliferating cells, a monoclonal antibody against Ki67 (Dako,Carpinteria, CA) was used at a 1:25 dilution. Keratinocyte differentiation wasevaluated using a transglutaminase antibody (Neomarkers, Fremont, CA) at a1:50 dilution. Primary antibodies were followed by rhodamine red donkeyantimouse secondary antibodies at a 1:100 dilution (Jackson Immunoresearch,West Grove, PA) or fluorescein-labeled donkey antimouse secondary antibod-ies (Jackson Immunoresearch) at a 1:2000 dilution, respectively.

For simultaneous detection of centrosomes and chromosome 11, cells werefixed in 4% paraformaldehyde for 10 min and permeabilized with 2% TritonX-100 for 20 min, both at room temperature. Cells were then denatured in 70%formamide/23SSC for 5 min at 72°C and stained for the pericentriolar markerg-tubulin followed by incubation with a chromosome 11a-satellite FISHprobe (Vysis, Downers Grove, IL) overnight at 37°C as described previ-ously (8).

Received 12/1/00; accepted 1/31/01.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant CA66980 (to K. M.). S. D. is supported by a postdoctoralfellowship from the Deutsche Forschungsgemeinschaft (Du 343/1-1). A. D. is supportedby a fellowship from the Dr. Mildred Scheel Stiftung. K. M. is a Ludwig Scholar.

2 To whom requests for reprints should be addressed, at Department of Pathology andHarvard Center for Cancer Biology, Harvard Medical School, 200 Longwood Avenue,Boston, MA 02115-5701. Phone: (617) 432-2878; Fax: (617) 432-0426; E-mail:karl_munger@hms.harvard.edu.

3 The abbreviations used are: HPV, human papillomavirus; NHK, normal humankeratinocyte; GFP, green fluorescent protein; pRB, retinoblastoma protein; FISH, fluo-rescencein situ hybridization.

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Apoptotic cells were visualized using the TACS 2 TdT-DABin situ apo-ptosis detection kit (Trevigen, Gaithersburg, MD) according to the manufac-turer’s instructions.

Detection of senescence-associatedb-galactosidase activity (pH 6.0) wasperformed as described previously (13).

Statistical Methods. Student’s two-tailedt test for independent sampleswas used wherever applicable to ascertain the statistical significance of thedifferences observed. Mean percentage and SE of at least three independentexperiments (at least 100 cells evaluated per experiment) are given unlessindicated otherwise.

Results

HPV-16 E7 Induces Abnormal Centrosome Numbers beforeExtensive Nuclear Abnormalities. We have shown previously (8)that abnormal centrosome numbers are increased in high-risk HPVE6- and/or E7-expressing cells. Whereas HPV E7 expression led to arapid increase of centrosome numbers, centrosome abnormalities inHPV E6-expressing cells were only observed after several weeks ofculture. For HPV-16 E7-induced centrosomal abnormalities to drivegenomic destabilization, these changes should develop early duringneoplastic progression in cells that do not yet display the typical

morphological alterations seen in tumor cells. These changes com-prise nuclear atypia, which is an important marker for the diagnosis ofcervical neoplasia as well as other malignancies (14). Nuclear abnor-malities can show different degrees of severity beginning with nuclearenlargement and irregular size and shape, but more advanced changesfrequently include the formation of multiple irregular nuclei. Toaddress the question of whether HPV-16 E6 or E7 oncogene-inducedcentrosome abnormalities represent early events during neoplasticprogression, centrosome numbers were assessed in NHKs displayinga single nucleus with no other signs of atypia. Cells that stably expresshigh-risk or low-risk E6 and/or E7 were compared with LXSN vector-infected control populations using the pericentriolar markerg-tubulin(Ref. 15; Fig. 1,A–D). Centrosome numbers exceeding two per cell(Fig. 1C) were considered abnormal.

Expression of HPV-16 E7 resulted in an increase of the proportionof mononucleated cells with abnormal centrosome numbers (Fig. 1D)to 6.8%. This represents a 3.4-fold increase (P # 0.05) compared tomatched controls (2%; Fig. 1,B and D). In NHKs expressing bothHPV-16 E6 and E7, 8.9% (4.5-fold increase;P # 0.005) of mono-nucleated cells displayed abnormal centrosomes (Fig. 1D). In contrast,

Fig. 1. A, immunoblot detection of HPV-16 E7 protein inNHKs with stable expression of this protein (top). Immunoblotanalysis revealed decreased levels of p53 in HPV-16 E6-expressing NHKs (bottom).B, normal centrosomes (arrowhead)in a LXSN-infected control cell.C, abnormal centrosome num-ber (arrowhead) in a HPV-16 E7-expressing mononucleatedcell. Centrosomes were visualized by immunofluorescence us-ing a g-tubulin antibody. Nuclei were counterstained with theDNA dye Hoechst 33258.D, quantitation of centrosome abnor-malities of cells showing a single nucleus in NHK populationsexpressing HPV-6 E6 or E7 or HPV-16 E6 and/or E7.E, normalcentrosomes and normal chromosome 11 copy number in aLXSN-infected control cell.F, three centrosomes in a mono-nucleated cell with normal chromosome 11 copy number. Cen-trosomes were visualized using immunofluorescence againstg-tubulin (red). Chromosome 11 copy numbers were visualizedby FISH using ana-satellite centromeric probe (green). Nucleiwere counterstained with the DNA dye Hoechst 33258.G,number of cells with abnormal centrosome numbers in mono-nucleated cells exhibiting two copies of chromosome 11.H,normal centriole number and colocalization of centrin-GFP(green) withg-tubulin (red) in a control cell of a U2OS clonestably expressing centrin-GFP.I, excessive synthesis of centri-oles (green) in U2OS/centrin-GFP cells stably expressingHPV-16 E7.g-Tubulin (red) colocalizes with three individualcentrioles. Nuclei were counterstained with the DNA dye Ho-echst 33258.J, quantitation of abnormal centriole numbers (.4centrioles/cell) in empty vector-infected controls and HPV-16E7-expressing U2OS/centrin-GFP cells.K, quantitation of ab-normal centriole numbers (.4 centrioles/cell) in controlsversusU2OS/centrin-GFP cells exhibiting one or twog-tubulin-posi-tive dots.

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expression of HPV-16 E6 or low-risk HPV-6 E6 and E7 had nosignificant effect on abnormal centrosome numbers in mononucleatedNHKs (Fig. 1D). We conclude that expression of HPV-16 E7 causesabnormal centrosome numbers in cells lacking morphological signs ofextensive nuclear abnormalities.

HPV-16 E7 Induces Centrosome Abnormalities in Mononucle-ated Cells with Normal Chromosome 11 Numbers.HPV-16 E7oncoprotein expression induces abnormal centrosome numbers incells lacking extensive nuclear atypia (Fig. 1,C andD). To rule outthe possibility that these cells are not polyploid or aneuploid undermaintenance of a mononucleated phenotype, we analyzed cells simul-taneously for centrosome abnormalities and copy numbers of chro-mosome 11 (Fig. 1,E–G) using a combination of immunofluores-cence and FISH. Copy number variability of chromosome 11 has beenreported previously (8) to increase early during the development ofgenomic instability in NHKs expressing high-risk HPV oncoproteins.We found abnormal centrosome numbers in 6.4% of mononucleatedcells expressing HPV-16 E7 and exhibiting a normal chromosome 11copy number (Fig. 1,F and G). In contrast, LXSN-infected controlcells and cells expressing HPV-16 E6 showed abnormal centrosomenumbers in only 0.63% and 0.62% of mononucleated cells withnormal chromosome 11 copy number, respectively (Fig. 1,E andG).This finding further supports the notion that expression of HPV-16 E7promotes abnormal centrosome duplication before the establishmentof extensive nuclear abnormalities.

HPV-16 E7 Increases Centriole Synthesis.Because these resultssuggest that HPV-16 E7 affects centrosome homeostasis as an earlyevent during neoplastic progression, we next sought to determinewhether this function of E7 is associated with accelerated centrosomesynthesis. To address this question, we manipulated the U2OS humanosteosarcoma cell line to stably express a centrin-GFP plasmid (kindlyprovided by M. Bornens; Ref. 12; Fig. 1,H–K). Centrin is aMr 20,000protein that associates with individual centrioles that form the corecomponents of centrosomes (12). U2OS/centrin-GFP cells were thenengineered to express HPV-16 E7 or empty vector used as negativecontrol. We found a 3.3-fold increase of the proportion of cells withabnormal centriole numbers from 6.3% in controls to 20.9% inHPV-16 E7-expressing cells (P# 0.005; Fig. 1J). According to arecently published study (12),g-tubulin-positive centrosomal struc-tures in a cell can contain one or two centrioles. To determine whetherexpression of HPV-16 E7 can induce an abnormal production ofcentrioles withing-tubulin-positive structures, we simultaneously an-alyzed cells for centriole numbers andg-tubulin expression (Fig. 1,H,I, and K). We found that in cells exhibiting a normal number ofg-tubulin dots (i.e.,1 or 2 dots/cell), 10.6% of HPV-16 E7-expressingcells showed an abnormal centriole number (.4 centrioles/cell; Fig.1K). This is a 2.5-fold increase compared to only 4.2% in control cells(P # 0.05). These data show that HPV-16 E7 induces excessivesynthesis of centrioles even in the presence of a normal number ofg-tubulin positive structures.

Expression of HPV-16 E6 Oncoprotein in Primary HumanKeratinocytes Is Associated with Nuclear Abnormalities and Ac-cumulation of Centrosomes.Expression of HPV-16 E6 in primaryhuman cells results in abnormal centrosome numbers only after aprolonged time interval. We sought to determine whether centrosomeabnormalities in those cells develop together with other cellular al-terations, particularly nuclear abnormalities. An increased proportionof NHKs expressing HPV-16 E6 alone or in combination withHPV-16 E7 displayed advanced nuclear atypia, namely multinucle-ation ($3 nuclei/cell). This alteration of nuclear morphology in thepresence of HPV-16 E6 is illustrated in Fig. 2.

We next quantitated these abnormalities and found that in theLXSN control virus-infected NHKs, cells with multiple nuclei repre-

sented 0.2% of all cells (Fig. 3A). In high-risk HPV-16 E6-expressingNHKs, the percentage of multinucleated cells, however, was increased5.5-fold to 1.1% (P# 0.05; Fig. 3A). In contrast, in HPV-16 E7-expressing cells, the percentage of cells with advanced nuclear atypiawas not increased (0.3%; Fig. 3A). Cells that like cervical cancersexpressed both HPV-16 E6 and E7 showed a significant 10.5-foldincrease of multinucleated cells to 2.1% compared to control cells(P # 0.005; Fig. 3A). NHKs infected with low-risk HPV-6 E6- orE7-expressing constructs showed similar levels of nuclear alterations(0.2% and 0.1%, respectively; Fig. 3A) compared to controls. The vastmajority of cells with multiple nuclei also showed other signs ofatypia,i.e., nuclear enlargement and irregular nuclear size and shape(Figs. 2 and 3B).

We next determined the number of centrosomes in cells withabnormally increased numbers of nuclei (Fig. 3B). Because of the lowfrequency of these cells in the absence of E6, we analyzed HPV-16E6- and HPV-16 E6/E7-expressing NHK populations only (Fig. 3,BandC). We found that more than 90% of cells with multiple nucleidisplayed abnormal centrosome numbers (Fig. 3C). There was atendency toward high centrosome numbers in cells with the mostdramatic nuclear abnormalities.

We next examined whether these cells were viable or rather repre-sented cells prone to undergo apoptotic cell death, replicative senes-cence, or differentiation. For this purpose, cells from NHK popula-tions expressing control virus or HPV-16 E6 were costained forcentrosomes and the proliferation-associated marker Ki67 (Fig. 3D).We found that 41% of HPV-16 E6-expressing cells with multiplenuclei and abnormal centrosome numbers expressed Ki67, which wasin a similar range compared to the overall percentage of Ki67 posi-tivity in the HPV E6 population with abnormal centrosomes (38%). Incontrast, the overall proportion of Ki67-positive cells with centrosomeabnormalities in the control population was 6%.

Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nickend labeling staining revealed only occasional apoptotic cells (,1%)even in the multinucleated population (Fig. 3D). Replicative senes-cence was analyzed using senescence-associatedb-galactosidase ac-tivity as a marker, and approximately one-third of the multinucleatedcells showed weakb-galactosidase activity (Fig. 3D). Expression ofthe differentiation marker transglutaminase was not detectable in the

Fig. 2. Low-power view of age-matched cultures of primary human keratinocytesmanipulated to express LXSN control virus or HPV-16 E6 and/or E7. An increasednuclear atypia was observed in the presence of HPV-16 E6. Multinucleated cells ($3nuclei/cell) are indicated byarrowheads.

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majority of multinucleated cells of the HPV-16 E6-infected NHKpopulation (Fig. 3D).

These results demonstrate that in the presence of HPV-16 E6, thereis an increased proportion of cells that displays marked nuclear atypiaand accumulates abnormal centrosome as the nuclear atypia advances.These cells still replicate DNA but apparently are unable to undergocoordinated cell division, and a proportion of such cells eventuallyundergoes replicative senescence.

Discussion

Centrosome abnormalities associated with abnormal, multipolarspindle formation have been detected in a wide range of malignanttumors (7). However, it is a matter of controversy whether abnormalcentrosome numbers drive genomic instability or whether they havebeen accumulated during aberrant cell divisions in genetically unsta-ble cells. The first model predicts that centrosome abnormalitiesprecede other manifestations of genomic instability, whereas, accord-ing to the second model, centrosome abnormalities will be preferen-tially detected in abnormal cells.

We have shown previously (8) that expression of HPV-16 E6 andE7 oncoproteins in primary human keratinocytes leads to abnormalcentrosome numbers as well as centrosome-related mitotic defects. Inthis study, we correlated abnormal centrosome numbers to nuclearatypia in age-matched NHK populations, and we showed that expres-sion of HPV-16 E7 can induce abnormal centrosome numbers inmononucleated cells before the development of extensive morpholog-ical aberrations. We detected excessive centriole formation in thepresence of E7, a proportion of which acquires the pericentriolarcomponentg-tubulin, which is essential for the nucleation of micro-tubules (12). In these cells, it is very likely that upon cell division,abnormal mitotic spindle formation occurs (8) as a consequence ofabnormal centrosome numbers. Hence HPV E7 and presumably func-tionally related viral and cellular oncogenes can cause centrosome

abnormalities that give rise to chromosome missegregation and there-fore precede nuclear and genomic abnormalities. In contrast, centro-some abnormalities in HPV-16 E6-expressing cells are associatedwith marked nuclear atypia, namely, multiple irregular nuclei. Thevast majority of E6-expressing cells with abnormal centrosome num-bers also exhibit multiple nuclei (Fig. 3B). Hence, in HPV-16 E6-expressing cells, centrosomes accumulate in parallel with nucleardamage. Many multinucleated cells showed nuclear immunoreactivityfor the proliferation marker Ki67, indicating that these cells are viablebut apparently unable to undergo a regulated division. It has beenreported that multinucleated squamous cells in cervical lesions werehighly positive for HPV DNA (16), and such abnormal cells maytherefore play some role in the viral life cycle. Previous studies haveshown that cells lacking p53 and p21Cip1 can reenter S phase withoutcompleting mitosis and cytokinesis and develop abnormal centrosomenumbers (17). Because, in our experiments, multinucleated cells weremost prevalent in cells expressing HPV-16 E6, it is conceivable thatthe known ability of E6 to impair p53-dependent checkpoint controlmay enable cells to reenter S phase and accumulate abnormal numbersof centrosomes. The extent of nuclear abnormalities that is observedin multinucleated cells would predict that these cells are prone toundergo apoptotic cell death. However, multinucleated cells did notshow an increased rate of cell death, but some of them eventuallyundergo replicative senescence (Fig. 3D).

In summary, there are two distinct pathways for the development ofabnormal centrosome numbers in tumor cells. Here we show that theyare not mutually exclusive; in contrast, they cooperate. The firstpathway involves abnormal duplication of centrosomes that drivechromosome missegregation and lead to aneuploidy. Our results sup-port this hypothesis by demonstrating that high-risk HPV E7 thatdegrades pRB induces increased centriole synthesis, leading to abnor-mal centrosome numbers before the appearance of extensive nuclearabnormalities. In cells expressing the high-risk HPV E6 oncoprotein

Fig. 3. A, quantitation of cells displaying multiplenuclei ($3 nuclei/cell) in NHK populations expressingHPV-6 E6, HPV-6 E7, or HPV-16 E6 and/or E7.B,multiple irregular nuclei in a HPV-16 E6-expressingkeratinocyte showing an abnormal number of centro-somes (arrowhead). Centrosomes were visualized usingimmunofluorescence againstg-tubulin (red). Nucleiwere counterstained with the DNA dye Hoechst 33258.C, quantitation of centrosome abnormalities in NHKpopulations stably expressing HPV-16 E6 or HPV-16E6/E7. D, analysis of cells in controlsversusHPV-16E6-expressing NHK populations for proliferation [dou-ble immunofluorescence for centrosomes (g-tubulin,red)and Ki-67 (green)], apoptotic cell death (DNAin situendlabeling, counterstaining with methyl-green), replicativesenescence (b-galactosidase activity,blue), or differen-tiation (transglutaminase expression,red). Nuclei werevisualized using Hoechst 33258 DNA dye.

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that inactivates p53, we observed accumulation of abnormal centro-some numbers in parallel with nuclear atypia, primarily multinucle-ation. In this case, centrosome abnormalities may merely reflectgenomic instability and develop as a secondary phenomenon. Becausecervical cancers express both E6 and E7, both processes, abnormalcentrosome duplication and accumulation of abnormal centrosomenumbers, are active during high-risk HPV-associated carcinogenesis.This is consistent with our previous study that showed that E6 and E7oncoproteins cooperate in inducing these alterations (8). The HPV-16E7 oncoprotein acts as a driving force for centrosome-related mitoticdefects and genomic instability by inducing abnormal centriole syn-thesis, whereas expression of E6 increases the likelihood that suchabnormal cells will remain proliferatively active and thus accumulatenuclear abnormalities. Given the frequent inactivation of the majorcellular targets of the HPV oncoproteins, the p53 and pRB tumorsuppressor pathways in many tumors, these findings have generalimplications for the induction and propagation of genomic instabilityduring human carcinogenesis

Acknowledgments

We are grateful to M. Bornens for the centrin-GFP construct, D. Gallowayand V. Band for retroviral vectors, J. S. Lee and R. Mulligan for sharingreagents, and A. Bonni for critical reading of the manuscript and helpfulcomments.

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2001;61:2356-2360. Cancer Res   Stefan Duensing, Anette Duensing, Christopher P. Crum, et al.   Evolving Malignant PhenotypeAbnormal Centrosome Synthesis Is an Early Event in the Human Papillomavirus Type 16 E7 Oncoprotein-induced

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