The human papillomavirus (HPV) E7 protein is one of only two viral proteins that remain expressed in HPVassociated human cancers. HPV E7 proteins share structural and functional similarities with oncoproteins encoded by other small DNA tumor viruses such as adenovirus E1A and SV40 large tumor antigen. The HPV E7 protein plays an important role in the viral life cycle by subverting the tight link between cellular dierentiation and proliferation in normal epithelium, thus allowing the virus to replicate in dierentiating epithelial cells that would have normally withdrawn from the cell division cycle. The transforming activities of E7 largely re¯ect this important function. Oncogene (2001) 20, 7888 ± 7898.

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 ...

The steady-state level and metabolic half-life of retinoblastoma tumor suppressor protein pRB are decreased in cells that express high-risk human papillomavirus (HPV) E7 proteins. Here we show that pRB degradation is a direct activity of E7 and does not reflect a property of cell lines acquired during the selection process for E7 expression. An amino-terminal domain of E7 that does not directly contribute to pRB binding but is required for transformation is also necessary for E7-mediated pRB degradation. Treatment with inhibitors of the 26S proteasome not only blocks E7-mediated pRB degradation but also causes the stabilization of E7. Mutagenic analyses, however, reveal that the processes of proteasomal degradation of E7 and pRB are not linked processes. HPV type 16 E7 also targets the pRB-related proteins p107 and p130 for destabilization by a proteasome-dependent mechanism. Using the SAOS2 flat-cell assay as a biological indicator for pRB function, we demonstrate that pRB degradation, not solely binding, is important for the E7-induced inactivation of pRB.Human papillomaviruses (HPVs) are DNA viruses with small circular genomes that cause epithelial hyperplasias ranging from benign papillomas (warts) to premalignant lesions that can progress to squamous cell carcinomas (reviewed in reference 25). There are over 100 different HPV types, approximately 30 of which specifically infect anogenital tract mucosa. These HPVs are classified as low risk or high risk, depending on the clinical prognosis of the lesions they cause. Low-risk HPVs, such as HPV type 6 (HPV-6) and HPV-11, cause condylomata acuminata (genital warts), while high-risk HPVs, including HPV-16 and HPV-18, are associated with squamous intraepithelial lesions that can progress to cervical carcinomas. Integration of the HPV genome into a host cell chromosome is a frequent event during malignant progression and results in the consistent but dysregulated expression of the HPV E6 and E7 proteins (reviewed in reference 55).High-risk HPV E6 proteins target tumor suppressor protein p53 for ubiquitin-mediated proteasomal degradation by interacting with and reprogramming the E6-AP ubiquitin ligase (38, 39, 51). E6-mediated degradation of p53 compromises the ability of the host cell to engage cell cycle checkpoints and apoptotic responses (33). High-risk HPV E7 oncoproteins target retinoblastoma tumor suppressor protein pRB (19). Highrisk HPV E7 proteins interact with pRB at a higher efficiency than do low-risk HPV E7 proteins (22,34). Interaction of E7 with hypophosphorylated pRB causes the disruption of growth-suppressive pRB-E2F complexes (10), promoting the G 1 -S cell cycle transition. E7-mediated cellular transformation correlates with pRB binding (22); however, there are mutations in E7 that impair cellular transformation and immortalization without affecting pRB binding (4,8,20,28,35). Furthermore, the E7 protein of HPV-1a, a low-risk cutaneous HPV, can interact with pRB and transactivate E2F-dependent promoters with the same efficiency as can high-risk ...

Our aim was to evaluate the expression of transcription factors CCAAT/enhancer-binding protein-beta (C/EBPbeta) and C/EBP-homologous protein (CHOP) in the growth plate. Proximal tibial epiphyseal growth plates from ten 15-day-old Wistar rats were used. Additionally, anti-proliferating cell nuclear antigen (PCNA), anti-5-bromo-2'-deoxyuridine (BrdU) immunostaining, terminal transferase dUTP nick end-labelling (TUNEL) and nucleolar organiser region-associated proteins (AgNOR) techniques were peformed. The histological morphology of the growth plate from C/EBPbeta knock-out mice was also analysed. The normal growth plate showed that C/EBPbeta and CHOP factors are expressed both in the germinative/ upper proliferative and in the lower proliferative zones. Furthermore, BdrU+ and PCNA+ cells were present exclusively in the germinative and proliferative zones, while TUNEL+ and AgNOR+ cells were seen in all three zones of the growth plate. Acellular areas, hypocellularity, the increase in cell death and anomalies in the architecture of the cell columns were observed in the growth plates of C/EBPbeta (-/-) knockout mice. We suggest that C/EBPbeta and CHOP transcription factors may be key modulators participating in the chondrocyte differentiation process in the growth plate.