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

Department of Pathology and Harvard Center for Cancer Biology, Harvard Medical School, Boston, Massachusetts 02115 [S. D., K. M.], and Department of Pathology, Solid Tumor 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

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Genomic instability is a hallmark of malignant growth that frequently involves mitotic defects associated with centrosome abnormalities. However, the question of whether abnormal centrosomes cause genomic instability or develop secondary to other changes has not been conclusively resolved. Here we show that human papillomavirus (HPV)-16 E7 can induce abnormal centrosome synthesis before the development of extensive nuclear abnormalities. In contrast, expression of HPV-16 E6 is associated with marked nuclear atypia and concomitant accumulation of centrosomes. Our results demonstrate that HPV-16 E7-induced centrosome abnormalities represent an early event during neoplastic progression potentially driving genomic destabilization.

	Introduction

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The development of cervical cancer is tightly associated with infection by high-risk HPV³ types such as HPV-16 or HPV-18, whereas low-risk HPV types like HPV-6 are associated with benign genital warts. High-risk HPVs encode two oncoproteins, E6 and E7, which subvert crucial cellular regulatory nodes to reactivate and maintain DNA synthesis in the host cell. Whereas high-risk HPV E6 mediates the accelerated proteosomal degradation of the p53 tumor suppressor protein, E7 binds to and destabilizes the retinoblastoma tumor suppressor (pRB) and interferes with the cyclin-dependent kinase inhibitor p21^Cip1 (1 , 2) .

Progression from normal to malignant cervical epithelium during high-risk HPV-associated carcinogenesis is characterized by the development of genomic instability as manifested by abnormal chromosome numbers, i.e., aneuploidy (3) . The high-risk HPV E6 and E7 oncoproteins can independently induce genomic instability in normal human cells (4, 5, 6) . As in many cancers (7) , genomic instability in high-risk HPV-induced lesions is associated with centrosome abnormalities (8) . The centrosome is the major microtubule organizing center in interphase and mitotic cells and assures symmetry and bipolarity of the cell division process by duplicating precisely once before a cell division (9) . Centrosome abnormalities have been detected in a wide variety of tumors; however, the mechanistic significance of centrosome abnormalities for the induction of genomic instability has been debated. On one hand, it has been proposed that abnormal centrosome numbers may directly cause mitotic defects that lead to genomic instability. According to this model, centrosome abnormalities emerge early during neoplastic progression in cells that do not yet show other manifestations of genomic instability. Alternatively, centrosome abnormalities may simply reflect genomic instability and accumulate in parallel with other cellular abnormalities. These two models have different implications for the diagnostic and prognostic value of centrosome abnormalities in human tumors. We have shown previously (8) that high-risk HPV E6/E7 oncoproteins cooperate to induce centrosome abnormalities and mitotic defects. Here we address the question whether E6- and E7-induced centrosome abnormalities drive the process of genomic instability or merely reflect the accumulation of cellular alterations. Expression of HPV-16 E7 is associated with an abnormal synthesis of centrioles and induces abnormal centrosome numbers early during neoplastic progression in primary human epithelial cells that do not display extensive nuclear abnormalities. In contrast, in HPV-16 E6-expressing cells, centrosome abnormalities accumulate in cells with marked nuclear atypia. These results support the model that HPV-16 E7 causes mitotic abnormalities through dysregulation of centrosome homeostasis early during malignant progression and therefore represents a potential driving force for genomic destabilization.

	Materials and Methods

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Cell Culture and Retroviral Infections.
NHKs from neonatal foreskins were isolated and cultured as described previously (2) . For retroviral infection of NHKs, recombinant retrovirus LXSN or LXSN-based retroviral constructs expressing HPV-6 E6 or E7 or HPV-16 E6 or E7 were used (10) . For coinfection of HPV-16 E6-expressing NHKs with HPV-16 E7, a high-titer pBABE E7 retroviral vector was produced as described previously (11) .

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

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

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

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

Apoptotic cells were visualized using the TACS 2 TdT-DAB in situ apoptosis detection kit (Trevigen, Gaithersburg, MD) according to the manufacturer’s instructions.

Detection of senescence-associated ß-galactosidase activity (pH 6.0) was performed as described previously (13) .

Statistical Methods.
Student’s two-tailed t test for independent samples was used wherever applicable to ascertain the statistical significance of the differences observed. Mean percentage and SE of at least three independent experiments (at least 100 cells evaluated per experiment) are given unless indicated otherwise.

	Results

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HPV-16 E7 Induces Abnormal Centrosome Numbers before Extensive Nuclear Abnormalities.
We have shown previously (8) that abnormal centrosome numbers are increased in high-risk HPV E6- and/or E7-expressing cells. Whereas HPV E7 expression led to a rapid increase of centrosome numbers, centrosome abnormalities in HPV E6-expressing cells were only observed after several weeks of culture. For HPV-16 E7-induced centrosomal abnormalities to drive genomic destabilization, these changes should develop early during neoplastic progression in cells that do not yet display the typical morphological alterations seen in tumor cells. These changes comprise nuclear atypia, which is an important marker for the diagnosis of cervical neoplasia as well as other malignancies (14) . Nuclear abnormalities can show different degrees of severity beginning with nuclear enlargement and irregular size and shape, but more advanced changes frequently include the formation of multiple irregular nuclei. To address the question of whether HPV-16 E6 or E7 oncogene-induced centrosome abnormalities represent early events during neoplastic progression, centrosome numbers were assessed in NHKs displaying a single nucleus with no other signs of atypia. Cells that stably express high-risk or low-risk E6 and/or E7 were compared with LXSN vector-infected control populations using the pericentriolar marker -tubulin (Ref. 15 ; Fig. 1, A–D ). Centrosome numbers exceeding two per cell (Fig. 1C) were considered abnormal.

View larger version (56K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. A, immunoblot detection of HPV-16 E7 protein in NHKs with stable expression of this protein (top). Immunoblot analysis 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 number (arrowhead) in a HPV-16 E7-expressing mononucleated cell. Centrosomes were visualized by immunofluorescence using a -tubulin antibody. Nuclei were counterstained with the DNA dye Hoechst 33258. D, quantitation of centrosome abnormalities of cells showing a single nucleus in NHK populations expressing HPV-6 E6 or E7 or HPV-16 E6 and/or E7. E, normal centrosomes and normal chromosome 11 copy number in a LXSN-infected control cell. F, three centrosomes in a mononucleated cell with normal chromosome 11 copy number. Centrosomes were visualized using immunofluorescence against -tubulin (red). Chromosome 11 copy numbers were visualized by FISH using an -satellite centromeric probe (green). Nuclei were counterstained with the DNA dye Hoechst 33258. G, number of cells with abnormal centrosome numbers in mononucleated cells exhibiting two copies of chromosome 11. H, normal centriole number and colocalization of centrin-GFP (green) with -tubulin (red) in a control cell of a U2OS clone stably expressing centrin-GFP. I, excessive synthesis of centrioles (green) in U2OS/centrin-GFP cells stably expressing HPV-16 E7. -Tubulin (red) colocalizes with three individual centrioles. Nuclei were counterstained with the DNA dye Hoechst 33258. J, quantitation of abnormal centriole numbers (>4 centrioles/cell) in empty vector-infected controls and HPV-16 E7-expressing U2OS/centrin-GFP cells. K, quantitation of abnormal centriole numbers (>4 centrioles/cell) in controls versus U2OS/centrin-GFP cells exhibiting one or two -tubulin-positive dots.

HPV-16 E7 Induces Centrosome Abnormalities in Mononucleated Cells with Normal Chromosome 11 Numbers.
HPV-16 E7 oncoprotein expression induces abnormal centrosome numbers in cells lacking extensive nuclear atypia (Fig. 1, C and D) . To rule out the possibility that these cells are not polyploid or aneuploid under maintenance of a mononucleated phenotype, we analyzed cells simultaneously for centrosome abnormalities and copy numbers of chromosome 11 (Fig. 1, E–G) using a combination of immunofluorescence and FISH. Copy number variability of chromosome 11 has been reported previously (8) to increase early during the development of genomic instability in NHKs expressing high-risk HPV oncoproteins. We found abnormal centrosome numbers in 6.4% of mononucleated cells expressing HPV-16 E7 and exhibiting a normal chromosome 11 copy number (Fig. 1, F and G) . In contrast, LXSN-infected control cells and cells expressing HPV-16 E6 showed abnormal centrosome numbers in only 0.63% and 0.62% of mononucleated cells with normal chromosome 11 copy number, respectively (Fig. 1, E and G) . This finding further supports the notion that expression of HPV-16 E7 promotes abnormal centrosome duplication before the establishment of extensive nuclear abnormalities.

HPV-16 E7 Increases Centriole Synthesis.
Because these results suggest that HPV-16 E7 affects centrosome homeostasis as an early event during neoplastic progression, we next sought to determine whether this function of E7 is associated with accelerated centrosome synthesis. To address this question, we manipulated the U2OS human osteosarcoma cell line to stably express a centrin-GFP plasmid (kindly provided by M. Bornens; Ref. 12 ; Fig. 1, H–K ). Centrin is a M_r 20,000 protein that associates with individual centrioles that form the core components of centrosomes (12) . U2OS/centrin-GFP cells were then engineered to express HPV-16 E7 or empty vector used as negative control. We found a 3.3-fold increase of the proportion of cells with abnormal centriole numbers from 6.3% in controls to 20.9% in HPV-16 E7-expressing cells (P 0.005; Fig. 1J ). According to a recently published study (12) , -tubulin-positive centrosomal structures in a cell can contain one or two centrioles. To determine whether expression of HPV-16 E7 can induce an abnormal production of centrioles within -tubulin-positive structures, we simultaneously analyzed cells for centriole numbers and -tubulin expression (Fig. 1, H, I, and K) . We found that in cells exhibiting a normal number of -tubulin dots (i.e., 1 or 2 dots/cell), 10.6% of HPV-16 E7-expressing cells 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 excessive synthesis of centrioles even in the presence of a normal number of -tubulin positive structures.

Expression of HPV-16 E6 Oncoprotein in Primary Human Keratinocytes Is Associated with Nuclear Abnormalities and Accumulation of Centrosomes.
Expression of HPV-16 E6 in primary human cells results in abnormal centrosome numbers only after a prolonged time interval. We sought to determine whether centrosome abnormalities in those cells develop together with other cellular alterations, particularly nuclear abnormalities. An increased proportion of NHKs expressing HPV-16 E6 alone or in combination with HPV-16 E7 displayed advanced nuclear atypia, namely multinucleation (3 nuclei/cell). This alteration of nuclear morphology in the presence of HPV-16 E6 is illustrated in Fig. 2 .

View larger version (202K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Low-power view of age-matched cultures of primary human keratinocytes manipulated to express LXSN control virus or HPV-16 E6 and/or E7. An increased nuclear atypia was observed in the presence of HPV-16 E6. Multinucleated cells (3 nuclei/cell) are indicated by arrowheads.

View larger version (66K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. A, quantitation of cells displaying multiple nuclei (3 nuclei/cell) in NHK populations expressing HPV-6 E6, HPV-6 E7, or HPV-16 E6 and/or E7. B, multiple irregular nuclei in a HPV-16 E6-expressing keratinocyte showing an abnormal number of centrosomes (arrowhead). Centrosomes were visualized using immunofluorescence against -tubulin (red). Nuclei were counterstained with the DNA dye Hoechst 33258. C, quantitation of centrosome abnormalities in NHK populations stably expressing HPV-16 E6 or HPV-16 E6/E7. D, analysis of cells in controls versus HPV-16 E6-expressing NHK populations for proliferation [double immunofluorescence for centrosomes (-tubulin, red) and Ki-67 (green)], apoptotic cell death (DNA in situ end labeling, counterstaining with methyl-green), replicative senescence (ß-galactosidase activity, blue), or differentiation (transglutaminase expression, red). Nuclei were visualized using Hoechst 33258 DNA dye.

We next examined whether these cells were viable or rather represented cells prone to undergo apoptotic cell death, replicative senescence, or differentiation. For this purpose, cells from NHK populations expressing control virus or HPV-16 E6 were costained for centrosomes and the proliferation-associated marker Ki67 (Fig. 3D) . We found that 41% of HPV-16 E6-expressing cells with multiple nuclei and abnormal centrosome numbers expressed Ki67, which was in a similar range compared to the overall percentage of Ki67 positivity in the HPV E6 population with abnormal centrosomes (38%). In contrast, the overall proportion of Ki67-positive cells with centrosome abnormalities in the control population was 6%.

Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining revealed only occasional apoptotic cells (<1%) even in the multinucleated population (Fig. 3D) . Replicative senescence was analyzed using senescence-associated ß-galactosidase activity as a marker, and approximately one-third of the multinucleated cells showed weak ß-galactosidase activity (Fig. 3D) . Expression of the differentiation marker transglutaminase was not detectable in the majority of multinucleated cells of the HPV-16 E6-infected NHK population (Fig. 3D) .

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

	Discussion

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Centrosome abnormalities associated with abnormal, multipolar spindle formation have been detected in a wide range of malignant tumors (7) . However, it is a matter of controversy whether abnormal centrosome numbers drive genomic instability or whether they have been accumulated during aberrant cell divisions in genetically unstable cells. The first model predicts that centrosome abnormalities precede other manifestations of genomic instability, whereas, according to the second model, centrosome abnormalities will be preferentially detected in abnormal cells.

We have shown previously (8) that expression of HPV-16 E6 and E7 oncoproteins in primary human keratinocytes leads to abnormal centrosome numbers as well as centrosome-related mitotic defects. In this study, we correlated abnormal centrosome numbers to nuclear atypia in age-matched NHK populations, and we showed that expression of HPV-16 E7 can induce abnormal centrosome numbers in mononucleated cells before the development of extensive morphological aberrations. We detected excessive centriole formation in the presence of E7, a proportion of which acquires the pericentriolar component -tubulin, which is essential for the nucleation of microtubules (12) . In these cells, it is very likely that upon cell division, abnormal mitotic spindle formation occurs (8) as a consequence of abnormal centrosome numbers. Hence HPV E7 and presumably functionally related viral and cellular oncogenes can cause centrosome abnormalities that give rise to chromosome missegregation and therefore precede nuclear and genomic abnormalities. In contrast, centrosome abnormalities in HPV-16 E6-expressing cells are associated with marked nuclear atypia, namely, multiple irregular nuclei. The vast majority of E6-expressing cells with abnormal centrosome numbers also exhibit multiple nuclei (Fig. 3B) . Hence, in HPV-16 E6-expressing cells, centrosomes accumulate in parallel with nuclear damage. Many multinucleated cells showed nuclear immunoreactivity for the proliferation marker Ki67, indicating that these cells are viable but apparently unable to undergo a regulated division. It has been reported that multinucleated squamous cells in cervical lesions were highly positive for HPV DNA (16) , and such abnormal cells may therefore play some role in the viral life cycle. Previous studies have shown that cells lacking p53 and p21^Cip1 can reenter S phase without completing mitosis and cytokinesis and develop abnormal centrosome numbers (17) . Because, in our experiments, multinucleated cells were most prevalent in cells expressing HPV-16 E6, it is conceivable that the known ability of E6 to impair p53-dependent checkpoint control may enable cells to reenter S phase and accumulate abnormal numbers of centrosomes. The extent of nuclear abnormalities that is observed in multinucleated cells would predict that these cells are prone to undergo apoptotic cell death. However, multinucleated cells did not show an increased rate of cell death, but some of them eventually undergo replicative senescence (Fig. 3D) .

In summary, there are two distinct pathways for the development of abnormal centrosome numbers in tumor cells. Here we show that they are not mutually exclusive; in contrast, they cooperate. The first pathway involves abnormal duplication of centrosomes that drive chromosome missegregation and lead to aneuploidy. Our results support this hypothesis by demonstrating that high-risk HPV E7 that degrades pRB induces increased centriole synthesis, leading to abnormal centrosome numbers before the appearance of extensive nuclear abnormalities. In cells expressing the high-risk HPV E6 oncoprotein that inactivates p53, we observed accumulation of abnormal centrosome numbers in parallel with nuclear atypia, primarily multinucleation. In this case, centrosome abnormalities may merely reflect genomic instability and develop as a secondary phenomenon. Because cervical cancers express both E6 and E7, both processes, abnormal centrosome duplication and accumulation of abnormal centrosome numbers, are active during high-risk HPV-associated carcinogenesis. This is consistent with our previous study that showed that E6 and E7 oncoproteins cooperate in inducing these alterations (8) . The HPV-16 E7 oncoprotein acts as a driving force for centrosome-related mitotic defects and genomic instability by inducing abnormal centriole synthesis, whereas expression of E6 increases the likelihood that such abnormal cells will remain proliferatively active and thus accumulate nuclear abnormalities. Given the frequent inactivation of the major cellular targets of the HPV oncoproteins, the p53 and pRB tumor suppressor pathways in many tumors, these findings have general implications for the induction and propagation of genomic instability during human carcinogenesis

	ACKNOWLEDGMENTS

 
We are grateful to M. Bornens for the centrin-GFP construct, D. Galloway and V. Band for retroviral vectors, J. S. Lee and R. Mulligan for sharing reagents, and A. Bonni for critical reading of the manuscript and helpful comments.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 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 postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (Du 343/1-1). A. D. is supported by 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 and Harvard 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{at}hms.harvard.edu

³ The abbreviations used are: HPV, human papillomavirus; NHK, normal human keratinocyte; GFP, green fluorescent protein; pRB, retinoblastoma protein; FISH, fluorescence in situ hybridization.

Received 12/ 1/00. Accepted 1/31/01.

	REFERENCES

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