The E7 Oncoprotein of Human Papillomavirus Type 16 Stabilizes p53 through a Mechanism Independent of p19ARF

In more than 90% of cervical cancers, DNA from anogenital human papillomaviruses (HPVs) is present (3). The anogenital HPVs are divided into two types, the high-risk types found in cervical cancers (most frequently HPV type 16 [HPV-16] and HPV-18) and the low-risk types commonly found in genital warts (HPV-6 and HPV-11). Both types of HPVs express two oncogenes, E6 and E7. However, only the high-risk viruses encode E6 oncoproteins that efficiently inactivate the tumor suppressor protein p53 (12, 56). The E7 proteins from high-risk HPVs are highly effective at inactivating another tumor suppressor, the retinoblastoma susceptibility protein (pRb), whereas E7 proteins from low-risk HPVs are less effective (26, 48). The ability of HPVs to inactivate these two tumor suppressor proteins appears to contribute to their ability to increase the risk of cancer in experimental animals (21, 37, 50). HPVs infect squamous epithelia and replicate in differentiated keratinocytes (28). There is evidence that the functions of E6 and E7 that contribute to cancer are those that allow DNA replication to occur in postmitotic cells. For example, the E7 protein induces DNA synthesis in differentiated postmitotic human keratinocytes (10). Inactivation of pRb is part of the mechanism through which E7 promotes entry of resting cells into S phase (1). E7 liberates pRb from members of the E2F family of transcription factors, thereby allowing induction of genes encoding proteins that function during S phase (6, 47). E6 stimulates the degradation of p53, a protein required for the inhibition of DNA synthesis in senescing normal diploid human fibroblasts (NDFs) (19). The activity of p53 is also required to arrest cycling cells in the G1 phase in response to DNA damage (35). The ability of p53 to induce a G1 arrest in response to ionizing radiation is in part dependent on the induction of the p21 gene, whose product inhibits the cyclin-dependent kinases (CDKs) (14, 16). CDKs phosphorylate pRb and release it from the E2F transcription factor, allowing expression of genes whose products promote DNA synthesis. CDK activity is inhibited when p53 induces p21 expression in response to ionizing radiation (16). E6 eliminates the induction of p21 by p53, allowing CDKs to remain active in NDFs exposed to ionizing radiation (16). Thus, under certain circumstances, both E6 and E7 promote passage from G1 to S phase, a process that appears to be essential for viral replication in postmitotic keratinocytes. A lack of appropriate regulation of the transition from G1 to S may contribute to the development of cancer (reviewed in reference 58). Indeed, the functions of pRb and p53 are disrupted directly or indirectly in the majority of human cancers (58). In cells expressing E7 but not E6, the levels of pRb protein are reduced while the levels of p53 are increased (13, 30). A decrease in pRb is consistent with the idea that E7 stimulates the transition into S phase; however, the increase in the amount of p53 is counterintuitive because p53 can induce growth arrest and apoptosis (35, 69). It is not clear whether p53 is activated by E7. On one hand, Massimi and Banks (42) used p53-responsive transcriptional reporter assays to show that p53 function is inhibited in cells expressing HPV-16 E7. In agreement with these findings, Vaziri et al. (66) found that HPV-16 E7 could inhibit transcription of a luciferase reporter gene cloned downstream from the promoter of the p53-responsive p21 gene. Other data have indicated that p21 protein levels are increased in cells expressing E7 and that the majority of this increase can be accounted for by posttranscriptional events independent of p53 (29, 31). Thus, there is not strong evidence that stabilization of p53 by E7 results in a stimulation of transcription of p21. On the other hand, Thomas and Laimins (63) showed that HPV-16 E7 expression in keratinocytes results in overexpression of the MDM2 protein, which is the product of another gene induced by p53. In keratinocytes expressing E6 with E7, MDM2 protein levels were reduced, prompting Thomas and Laimins to hypothesize that E7 upregulates MDM2 by synergizing with factors that regulate p53 activity (63). In sum, it has not been established whether p53 is activated as a transcriptional regulator by E7. Recently, the E1A, c-myc, and E2F1 proteins have been shown to stabilize p53 and stimulate its transcriptional activation function (15, 71). Expression of E1A in mouse embryo fibroblasts (MEFs) appears to disrupt the interaction between p53 and its inhibitor, MDM2 (15). MDM2 directly blocks the ability of p53 to stimulate transcription (44, 49), and inhibition of the interaction between MDM2 and p53 results in activation of p53 (2). In addition, binding of MDM2 to p53 is required for the ability of MDM2, a ubiquitin ligase (27), to target p53 for degradation (25, 36). The ability of E1A to stabilize p53 appears to be dependent on the p19ARF protein (15), which inhibits the ability of MDM2 to ubiquitinate p53 (27). However, both c-myc and E2F1 elevate p53 levels in MEFs lacking p19ARF (71). E7 elevates p53 levels in NDFs (30) and expands the life span of these cells. In contrast, antibodies that block the interaction between MDM2 and p53 in NDFs cause an increase in p53 protein that results in arrest of cell division (2, 19). Therefore, we investigated whether E7 inhibited the regulation of p53 by MDM2 in NDFs.