Co-localization of the tumor-suppressor protein p53 and human papillomavirus E6 protein in human cervical carcinoma cell lines.

The loss of the tumor-suppressor activity of p53, either by mutation or by interaction with the human papillomavirus (HPV) E6 protein, is considered to be an important mechanism in the carcinogenesis of cervical cancer. We have studied the cytological distribution of these proteins in human cervical carcinoma cell lines using polyclonal anti-p53 and monoclonal anti-E6 antibodies. The antibody specificity was confirmed by immunoblot and immunocompetition analyses. The intracellular localization of p53 and E6 was detected using the techniques of conventional and three-dimensional confocal microscopy. In the HPV-18 or -16 integrated cell lines, HeLa, CaSki and SiHa, viral oncoprotein E6 and endogenous tumor-suppressor protein, p53, were observed by immunofluorescence in the cytoplasm; p53 also had a weak punctate staining in the nuclei of HeLa and CaSki cells. In the HPV-negative cervical carcinoma cell lines, C-33A and HT-3, which have mutated p53, p53 was localized predominantly to the nucleus, with C-33A cells having elevated levels of p53 compared with the other cell lines. High spatial resolution imaging, using confocal microscopy, was performed on the cells after double fluorescence staining for p53 (fluorescein) and E6 (rhodamine). The images showed that both p53 and E6 had similar cytoplasmic distributions, which implied that these two proteins may exist as a cytoplasmic complex. To substantiate this implication, fluorescence resonance energy transfer microscopy was performed, which provided direct evidence of a close association between p53 and E6 within individual HeLa cells. The results from this study support the theory that p53 protein binds HPV-16/18 E6 protein in the cell cytoplasm, thus preventing p53 from exerting its tumor-suppressor function in the nucleus. Hence, inactivation of wild-type p53 by p53-E6 complex formation in cervical cancer may be a critical step in malignant transformation.