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.