The ubiquitin-protein ligase E6-AP is utilized by the E6 oncoprotein of human papillomaviruses (HPVs) associated with cervical cancer to target the tumor suppressor p53 for degradation. Here, we report that downregulation of E6-AP expression by RNA interference results in both the accumulation of p53 and growth suppression of the HPV-positive cervical cancer cell lines HeLa and SiHa. In addition, HeLa cells, in which p53 expression was suppressed by RNA interference, are significantly less sensitive to the downregulation of E6-AP expression with respect to growth suppression than parental HeLa cells. These data indicate that the antigrowth-suppressive properties of E6-AP in HPV-positive cells depend on its ability to induce p53 degradation.Modification of proteins by the covalent attachment of ubiquitin or ubiquitin-related proteins is involved in the regulation of many cellular and viral processes (3, 10, 23). Remarkably, not only do viral proteins represent substrates for such proteinprotein modification systems, but some are also intrinsically involved in the conjugation of ubiquitin (ubiquitination) to cellular proteins, thereby redirecting the ubiquitin conjugation system for viral purposes (3). A prominent example for viral proteins associated with the ubiquitin-conjugation system is provided by the E6 oncoprotein of high-risk human papillomaviruses (HPVs) that are etiologically associated with cervical cancer and other malignant lesions of the anogenital tract. The E6 oncoprotein binds to the cellular ubiquitin-protein ligase E6-AP and utilizes E6-AP to target the tumor suppressor protein p53 for ubiquitination and subsequent proteasomemediated degradation (13, 24). Furthermore, E6 has been reported to target additional proteins, including E6TP1, hScrib, hDlg, and Bak for ubiquitination and degradation in an E6-AP-dependent or E6-AP-independent manner (18, 25).Continuous expression of E6 and E7, the two major HPV oncoproteins, is required for the maintenance of the transformed phenotype of cervical cancer cell lines (28). On the functional level, E6 has p53-dependent as well as p53-independent antiapoptotic properties (22). Indeed, interference with E6 expression or E6 activity results in the induction of apoptosis in HPV-positive cells, which is accompanied by a significant increase in p53 levels (6, 7). Similarly, the downregulation of E6-AP expression by antisense approaches or overexpression of a catalytically inactive E6-AP mutant results in the accumulation of p53 in HPV-positive cells but not in HPVnegative cells (4, 26). Moreover, ribozyme-mediated reduction of E6-AP expression enhances the apoptotic response of HeLa cells, an HPV-18-positive cell line, to the DNA damage-inducing drug mitomycin C (16). However, since E6-AP has been implicated in E6-mediated degradation of proteins other than p53 (e.g., E6TP1 and hScrib) (9, 21), it remains unclear if this apoptosis-enhancing effect is directly linked to the ability of E6-AP to target p53 for degradation in the presence of E6.To determine if the pr...