COLOCALIZATION 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 o r by interaction with the human papillomavirus (HPV) E6 protein, is co nsidered to be an important mechanism in the carcinogenesis of cervica l cancer. We have studied the cytological distribution of these protei ns in human cervical carcinoma cell lines using polyclonal anti-p53 an d monoclonal anti-E6 antibodies. The antibody specificity was confirme d by immunoblot and immunocompetition analyses. The intracellular loca lization of p53 and E6 was detected using the techniques of convention al and three-dimensional confocal microscopy. In the HPV-18 or -16 int egrated cell lines, HeLa, CaSki and SiHa, viral oncoprotein E6 and end ogenous tumor-suppressor protein, p53, were observed by immunofluoresc ence in the cytoplasm; p53 also had a weak punctate staining in the nu clei of HeLa and CaSki cells. In the HPV-negative cervical carcinoma c ell lines, C-33A and HT-3, which have mutated p53, p53 was localized p redominantly to the nucleus, with C-33A cells having elevated levels o f p53 compared with the other cell lines. High spatial resolution imag ing, using confocal microscopy, was performed on the cells after doubl e fluorescence staining for p53 (fluorescein) and E6 (rhodamine). The images showed that both p53 and E6 had similar cytoplasmic distributio ns, which implied that these two proteins may exist as a cytoplasmic c omplex. To substantiate this implication, fluorescence resonance energ y 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 e xerting its tumor-suppressor function in the nucleus. Hence, inactivat ion of wild-type p53 by p53-E6 complex formation in cervical cancer ma y be a critical step in malignant transformation.