Loss of genomic integrity is a defining feature of many human malignancies, including human papillomavirus (HPV)-associated preinvasive and invasive genital squamous lesions. Here we show that aberrant mitotic spindle pole formation caused by abnormal centrosome numbers represents an important mechanism in accounting for numeric chromosomal alterations in HPV-associated carcinogenesis. Similar to what we found in histopathological specimens, HPV-16 E6 and E7 oncoproteins cooperate to induce abnormal centrosome numbers, aberrant mitotic spindle pole formation, and genomic instability. The low-risk HPV-6 E6 and E7 proteins did not induce such abnormalities. Whereas the HPV-16 E6 oncoprotein has no immediate effects on centrosome numbers, HPV-16 E7 rapidly induces abnormal centrosome duplication. Thus our results suggest a model whereby HPV-16 E7 induces centrosome-related mitotic disturbances that are potentiated by HPV-16 E6.H uman papillomaviruses (HPVs) are small epitheliotropic DNA viruses involved in the etiology of several human malignancies. At least 90% of all cervical carcinomas are associated with infections by ''high-risk'' HPV types such as HPV-16 and -18. The majority of these cancers contain HPV DNA integrated into the host cell genome and express only two viral genes, E6 and E7, both of which encode oncoproteins (1). Both HPV-immortalized cells and high-risk HPV-associated cervical neoplasias, including early precursor lesions, display genomic instability, which is absent in lesions caused by low-risk HPVs (2-6). Induction of genomic plasticity, therefore, constitutes an early and central event in HPV-associated carcinogenesis and may contribute to the integration of HPV DNA into the host genome (7). However, it is not known in detail how HPV E6 and E7 interfere with genomic integrity. HPV E6 and E7 play distinct roles in this process by targeting different pathways (5). Whereas E6 may promote genetic instability by inactivating the tumor suppressor and cell cycle checkpoint protein p53 (5, 8), the mechanism by which E7 subverts the integrity of the host cell genome (5, 9) and whether this function depends on its ability to inactivate the pRB tumor suppressor protein (10, 11) have not been determined.The centrosome is a cytoplasmic organelle consisting of a pair of centrioles surrounded by a pericentriolar matrix. Each cell contains one or, before a cell division, two centrosomes. During mitosis, the two centrosomes form the poles of a bipolar mitotic spindle, a function that is essential for accurate chromosome segregation. Centrosomes undergo duplication precisely once before cell division. Recent reports have revealed that this process is linked to the cell division cycle via cyclin-dependent kinase 2 (cdk2) activity that couples centriole duplication to the onset of DNA replication at the G 1 ͞S transition (12-14).Various human malignancies exhibit centrosome abnormalities that contribute to defective mitotic spindle pole formation, thus causing chromosome missegregation and genetic instability ...

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Dysregulation of the centrosome duplication cycle has been implicated in tumorigenesis. Our previous work has shown that the human papillomavirus type 16 (HPV-16) E7 oncoprotein rapidly induces aberrant centrosome and centriole duplication in normal human cells. We report here that HPV E7-induced abnormal centriole duplication is specifically abrogated by a small molecule CDK inhibitor, indirubin-3 0 -oxime (IO), but not a kinase-inactive derivative. Importantly, normal centriole duplication was not markedly affected by IO, and the inhibitory effects were observed at concentrations that did not affect the G1/S transition of the cell division cycle. Depletion of CDK2 by siRNA similarly abrogated HPV E7-induced abnormal centrosome duplication and ectopic expression of CDK2 in combination with cyclin E or cyclin A could rescue the inhibitory effect of IO. IO treatment also reduced the steady-state level of aneuploid cells in HPV-16 E7-expressing cell populations. Our results suggest that cyclin/CDK2 activity is critically involved in abnormal centrosome duplication induced by HPV-16 E7 oncoprotein expression, but may be dispensable for normal centrosome duplication and cell cycle progression.