Failure of HPV E6 to rapidly degrade p53 in human HeLa×PNET cell hybrids

Abstract

The ability of the E6 protein from high risk human papillomaviruses (HPVs) to degrade p53 via the ubiquitin pathway plays a major role in the development of cervical carcinomas. We have previously generated cell hybrids between a p53 null peripheral neuroepithelioma (PNET) cell line and a cervical carcinoma HeLa cell line which exhibits ecient E6-mediated degradation of p53. All of the resulting hybrids expressed HPV 18 E6 from the HeLa parent and some of the hybrids additionally expressed HPV 16 E6. Surprisingly, in spite of abundant E6 expression, the hybrids expressed relatively high steady-state levels of the wild-type p53 protein. We then examined the hybrids to determine whether other components of the E6-mediated degradation pathway were missing or nonfunctional. Speci®cally, we determined that the E6-associated protein (E6-AP), essential for E6-mediated degradation, was expressed. We further veri®ed that these hybrids had a functional ubiquitination pathway, which suggests that this phenomenon is not due to a general defect in this pathway. We therefore conclude that other unidenti®ed, possibly cellspeci®c factors can play a role in the E6-mediated degradative process and may act to inhibit this process.

Comparison of human papillomavirus type 18 (HPV-18) E6-mediated degradation of p53 in vitro and in vivo reveals significant differences based on p53 structure and cell type but little difference with respect to mutants of HPV-18 E6.

“…These results clearly demonstrate, however, that the cellular environment can have a profound effect on the susceptibility of a protein to E6-mediated degradation. This conclusion is also supported by a recent study where a hybrid of p53-null peripheral neuroepithelial cells and HeLa cells expressed high levels of p53 protein, despite retaining HPV-18 E6 expression (Isaacs et al, 1997).…”

Mitochondrial DNA determines the cellular response to cancer therapeutic agents

“…p53 was recently shown to control the expression of several genes involved in protecting cells from mitochondria mediated apoptosis (Polyak et al, 1997). Since HeLa cells are de®cient in p53 function (Isaacs et al, 1997), our results also suggest that resistance to adriamycin and PDT is independent of p53 function. These results are further supported by Fisher et al (1999) who recently provided direct evidence that tumor cell sensitivity to PDT is independent of p53.…”

Interference of proteins involved in the cytoplasmic sequestration of p53 with human papillomavirus E6-mediated degradation

“…Logarithmically growing cells were grown to approximately 70% confluence, solubilized in NP-40 lysis buffer and quantitated as described previously [32]. For p53 western blot analysis, 25 µg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, blocked, incubated with the p53-specific antibody DO-1 (Santa Cruz Biotechnology, Santa Cruz, CA), and incubated with secondary antibody, as previously described.…”

“…We previously demonstrated high steady-state levels of p53 in HPV 16 E6-infected peripheral neuroepithelioma (PNET) × HeLa cell hybrids [32]. However, the protein is efficiently degraded in the HeLa parent as well as in p53-transfected PNET clonal cell lines [33].…”