The critical immortalizing activity of the human papillomavirus (HPV) type-16 E6 oncoprotein is to induce expression of hTERT, the catalytic and rate-limiting subunit of telomerase. Additionally, E6 binds to a cellular protein called E6-associated protein (E6-AP) to form an E3 ubiquitin ligase that targets p53 for proteasome-dependent degradation. Although telomerase induction and p53 degradation are separable and distinct functions of E6, binding of E6 to E6-AP strongly correlated with the induction of hTERT. Here, we demonstrate using shRNAs to reduce E6-AP expression that E6-AP is required for E6-mediated telomerase induction. A yeast two-hybrid screen to find new targets of the E6/E6-AP E3 ubiquitin ligase complex identified NFX1. Two isoforms of NFX1 were found: NFX1-123, which coactivated with c-Myc at the hTERT promoter, and NFX1-91, which repressed the hTERT promoter. NFX1-91 was highly ubiquitinated and destabilized in epithelial cells expressing E6. Furthermore, knockdown of NFX1-91 by shRNA resulted in derepression of the endogenous hTERT promoter and elevated levels of telomerase activity. We propose that the induction of telomerase by the HPV-16 E6/E6-AP complex involves targeting of NFX1-91, a newly identified repressor of telomerase, for ubiquitination and degradation.[Keywords: Telomerase; HPV; transcriptional repressor; ubiquitin; E6; E6-AP] Supplemental material is available at http://www.genesdev.org.

The transcription factor c-Myc is implicated in the pathogenesis of many cancers. Among the multiple functions of c-Myc, activation of hTert and other genes involved in cellular life span contributes to its role as an oncogene. However, the ability of c-Myc to directly immortalize human cells remains controversial. We show here that overexpression of c-Myc reproducibly immortalizes freshly isolated human foreskin fibroblasts. c-Myc -immortalized cells displayed no gross karyotypic abnormalities but consisted of an oligoclonal population, suggesting that additional events cooperated to achieve immortalization. Levels of p53 and p16 were increased, but both p53-dependent DNA damage response and growth arrest in response to p16 overexpression remained intact. A marked decrease in expression of the tumor suppressor ARF occurred in several independently established c-Myc -immortalized cell lines. Methylation-specific PCR showed that the ARF gene was methylated in immortalized but not early-passage c-Myc cells, whereas p16 was unmethylated in both cell populations. Restoration of ARF expression by treatment with a demethylating agent or overexpression by a retroviral vector coincided with inhibition of proliferation and senescence of c-Myc -immortalized cells. Our findings predict that epigenetic events play a significant role in human tumors that express high levels of c-Myc.

Nearly 20% of cancers worldwide have a component of their etiology that is due to infectious agents. In some cases, infection has an indirect effect, such as the immunosuppression caused by HIV or the inflammation caused by Helicobacter pylori, but in other cases, such as human papillomaviruses (HPVs), viral gene products persist in the cancer and directly promote neoplasia. Understanding the mechanisms by which the viral genes disrupt the checkpoints that normally protect cells from cancer will likely provide insights into cancers in which the underlying critical abnormalities are more difficult to discern.Both epidemiologic observations and molecular data firmly support a causal role for a group of HPVs in the etiology of virtually 100% of cervical carcinomas, as well as the majority of other anogenital cancers and a subset of head and neck cancers (Cogliano et al. 2005). Of these HPV types, HPV-16 DNA is found in more than 50% of tumors (Walboomers et al. 1999). Two viral genes, E6 and E7, are invariably retained and expressed in cervical cancers, and together E6 and E7 efficiently immortalize human epithelial cells. The E7 protein associates with the retinoblastoma (Rb) family of proteins through a LXCXE motif and promotes the ubiquitin-mediated degradation of Rb, p107, and p130 (Munger et al. 2001). Degradation of Rb is necessary but not sufficient for E7's role in cellular immortalization, which also requires sequences in the carboxy-terminal zinc-like finger (Helt and Galloway 2001). HPV-16 E6 associates with a cellular protein, E6AP, and together the complex functions as a ubiquitin ligase (Huibregtse et al. 1993). The p53 tumor suppressor is the best-studied target of E6/E6AP, and its degradation eliminates several checkpoints that normally maintain genetic stability (Kessis et al. 1993;Demers et al. 1994).Disruption of the Rb and p53 pathways is critical for transformation of many human cell types, but it is also essential to prevent telomere shortening (Hahn et al. 1999). In some strains of human fibroblasts, the introduction of hTERT, the catalytic subunit of telomerase, is sufficient for immortalization (Bodnar et al. 1998;Kiyono et al. 1998;Vaziri and Benchimol 1998;Wang et al. 1998). Telomerase may play additional roles in tumorigenic transformation, because hTERT was necessary for transformation of cells that maintained long telomeres by the ALT pathway (Stewart et al. 2002). Nearly all tumors and cells transformed in culture express hTERT at levels that provide sufficient telomerase activity to keep telomeres above a critically short level (Kim et al. 1994). Multiple mechanisms are likely responsible for regulation of telomerase activity, including changes in transcription factors (Xu et al. 2001), loss of transcriptional repressors (Horikawa et al. 1998;Ducrest et al. 2001;Lin and Elledge 2003), changes in chromatin structure (Takakura et al. 2001;Hou et al. 2002), and altered levels of telomere-binding proteins (van Steensel et al. 1998). Additionally, cancers may arise in stem cells in which ...

Supplementary Figures S1-S2 from Epigenetic Down-Regulation of ARF Expression Is a Selection Step in Immortalization of Human Fibroblasts by c-Myc

Supplementary Figures S1-S2 from Epigenetic Down-Regulation of ARF Expression Is a Selection Step in Immortalization of Human Fibroblasts by c-Myc