Complete switch from Mdm2 to human papillomavirus E6-mediated degradation of p53 in cervical cancer cells.

The E6 oncoprotein of human papillomaviruses (HPVs) that are associated with cervical cancer utilizes the cellular ubiquitin-protein ligase E6-AP to target the tumor suppressor p53 for degradation. In normal cells (i.e., in the absence of E6), p53 is also a target of the ubiquitin-proteasome pathway. Under these conditions, however, p53 degradation is mediated by Mdm2 rather than by E6-AP. Here we show in a mutational analysis that, surprisingly, the structural requirements of p53 to serve as a proteolytic substrate differ between E6 proteins derived from different HPV types and, as expected, between Mdm2 and E6 proteins in vitro and in vivo. Stable expression of such mutants in HPV-negative and HPV-positive cell lines demonstrates that in HPV-positive cancer cells, the E6-dependent pathway of p53 degradation is not only active but, moreover, is required for degradation of p53, whereas the Mdm2-dependent pathway is inactive. Because the p53 pathway was reported to be functional in HPV-positive cancer cells, this finding indicates clearly that the ability of the E6 oncoprotein to target p53 for degradation is required for the growth of HPV-positive cancer cells.

Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1.

The growth-suppressive properties of p53 are controlled by posttranslational modifications and by regulation of its turnover rate. Here we show that p53 can be modified in vitro and in vivo by conjugation to the small ubiquitin-like protein SUMO-1. A lysine residue at amino acid position 386 of p53 is required for this previously undescribed modification, strongly suggesting that this lysine residue serves as the major attachment site for SUMO-1. Unlike ubiquitin, attachment of SUMO-1 does not appear to target proteins for rapid degradation but rather, has been proposed to change the ability of the modified protein to interact with other cellular proteins. Accordingly, we provide evidence that conjugation of SUMO-1 to wild-type p53 results in an increased transactivation ability of p53. We suggest that posttranslational modification of p53 by SUMO-1 conjugation provides a novel mechanism to regulate p53 activity.

Replication of ONYX-015, a potential anticancer adenovirus, is independent of p53 status in tumor cells.

The 55-kDa E1B protein of adenovirus, which binds to and inactivates the tumor suppressor protein p53, is not expressed in the adenoviral mutant termed ONYX-015 (i.e., dl1520). It was reported that the mutant virus due to a deletion in E1B is able to replicate only in cells deficient for wild-type p53. Accordingly, dl1520 is currently being evaluated as a potential tool in the therapy of p53 deficient cancers. In contrast, we report here that dl1520 replicates independently of the p53 status in various tumor cell lines (U87, RKO, A549, H1299, and U373). In addition, the inhibition of p53-mediated transcriptional activation in wild-type p53 containing U2OS cells, by overexpression of a transdominant negative p53 mutant, did not render the cells permissive for dl1520 replication. Finally, we show that, depending on the multiplicity of infection, the deleted virus is able to replicate in and to kill primary human cells. Thus, the molecular basis for the growth differences of dl1520 within different cell types remains to be determined.

Characterization of sequence elements involved in p53 stability regulation reveals cell type dependence for p53 degradation.

The growth suppressive properties of the tumor suppressor protein p53 are activated upon DNA damage. The activation of p53 is reflected in increased p53 levels which are, at least in part, the result of an extended half-life of the protein. Although this suggests that stabilization of p53 is an intrinsic feature of p53 activation, the mechanisms involved in p53 degradation and stabilization are poorly understood. Here we report on the identification of an internal deletion mutant of wild-type p53, termed delta62-96, which can be stably expressed in various cell lines. This deletion mutant has a turnover rate similar to wild-type p53 and its stability is upregulated by treatment with UV light. In cell lines that express endogenous mutant or no p53, however, delta62-96 appears to be stable, strongly indicating that these cell lines have lost the ability to degrade p53. Further characterization of delta62-96 by mutational analyses defines sequence and structural requirements for p53 degradation and indicates that none of the known p53 phosphorylation sites is essential with respect to p53 stability regulation upon UV-irradiation.