Expression of the bovine papillomavirus E2 regulatory protein in human cervical carcinoma cell lines repressed expression of the resident human papillomavirus E6 and E7 oncogenes and within a few days caused essentially all of the cells to synchronously display numerous phenotypic markers characteristic of cells undergoing replicative senescence. This process was accompanied by marked but in some cases transient alterations in the expression of cell cycle regulatory proteins and by decreased telomerase activity. We propose that the human papillomavirus E6 and E7 proteins actively prevent senescence from occurring in cervical carcinoma cells, and that once viral oncogene expression is extinguished, the senescence program is rapidly executed. Activation of endogenous senescence pathways in cancer cells may represent an alternative approach to treat human cancers. Cancer is thought to be caused by the stepwise accumulation of genetic changes, resulting in progressive phenotypic abnormality. Infection of cervical keratinocytes with a highrisk human papillomavirus (HPV) such as HPV18 is the initiating event in the great majority of cervical carcinomas (1). The HPV E6 and E7 proteins are continuously expressed in cervical carcinoma cells and seem to play a role in tumor development and in maintenance of the malignant phenotype (1). The role of the E6 and E7 proteins in the progression from normal keratinocytes to transformed cells has been studied in cell culture. Normal human keratinocytes can undergo a limited number of cell divisions in vitro and then stop proliferating, a phenomenon referred to as replicative senescence (2-4). Telomere length progressively shortens during cell passage (5, 6), and it has been proposed that once the length of one or more telomeres falls below a critical threshold, an active genetic program is mobilized that culminates in senescence (7,8). Cultured keratinocytes can escape senescence and become immortalized by inactivation of the retinoblastoma tumor suppressor pathway and activation of telomerase, the enzyme responsible for maintaining telomere length (9, 10). Expression of the HPV E6 and E7 proteins also can immortalize keratinocytes (11,12), an activity that is consistent with the known biochemical activities of these proteins. The E7 protein neutralizes the retinoblastoma tumor suppressor pathway, and the E6 protein stimulates telomerase activity in keratinocytes (1,6,13). Although the mechanistic basis for this effect on telomerase activity is not known, this activity seems crucial for immortalization because E6 mutants defective for telomerase activation are immortalization-defective (10). In addition, host-cell mutations are required for HPVimmortalized keratinocytes to become tumorigenic and to acquire other malignant characteristics, such as metastatic potential (9, 14 -20). The E6 and E7 proteins facilitate the acquisition of these mutations by causing accelerated degradation of p53 and p105 Rb , respectively, resulting in abrogation of DNA damage checkpoint control, elevat...

The function of the human papillomavirus (HPV) E6 protein that is most clearly linked to carcinogenesis is the targeted degradation of p53, which is dependent on the E6AP ubiquitin ligase. Additional functions have been attributed to E6, including the stimulation of telomerase activity and the targeted degradation of other cellular proteins, but in most cases it is unclear whether these activities are also E6AP dependent. While E6 clearly influences the transcriptional program of HPV-positive cell lines through the inactivation of p53, it has been shown that at least a subset of its p53-independent functions are also reflected in the transcriptional program. For this study, we have determined the extent to which E6AP is involved in mediating the set of E6 functions that impact on the global transcriptional program of HPV-positive cell lines. The transcriptional profiles of ϳ31,000 genes were characterized for three cell lines (HeLa, Caski, and SiHa cells) after small interfering RNA (siRNA)-mediated silencing of E6 or E6AP. We found that E6 and E6AP siRNAs elicited nearly identical alterations in the transcriptional profile of each cell line. Some of the expression alterations were apparent secondary effects of p53 stabilization, while the basis of most other changes was not reconcilable with previously proposed E6 functions. While expression changes of the TERT gene (telomerase catalytic subunit) were not revealed by the array, telomerase repeat amplification protocol assays showed that both E6 and E6AP knockouts resulted in a suppression of telomerase activity. Together, these results suggest that E6AP mediates a broad spectrum of E6 functions, including virtually all functions that impact on the transcriptional program of HPV-positive cell lines.Human papillomaviruses (HPVs) are small DNA tumor viruses that infect cutaneous or mucosal epithelial cells. Approximately 30 HPV types are sexually transmitted and can be further categorized according to their associations with malignancies (65). Low-risk HPV types (e.g., types 6 and 11) are associated with benign lesions, while DNAs of high-risk HPV types (e.g., types 16, 18, 33, and 39) are found in Ͼ95% of all cervical cancers, strongly suggesting that HPV is a causative agent of this disease (65). The only two viral proteins that are generally expressed in HPV-positive cervical cancers, E6 and E7, have been shown to be necessary and sufficient to immortalize human foreskin keratinocytes, further suggesting that they are cooperating oncoproteins (21, 41). HPV E7 interacts with and inactivates the retinoblastoma protein (pRb) and the pRb-related proteins p107 and p130 (40). E7 inactivation of pRb activates E2F transcription factors, which results in increased transcription of E2F-responsive genes and S-phase cell cycle progression. While several other E7-interacting proteins have been identified (including histone deacetylase [38], p21, TATA-binding protein, and TAF110 [40]), results from animal studies indicate that the major biologic activities of E7 are related ...