Liang X H, Volkmann M, Klein R, Herman B, Lockett S J
Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill 27599.
Oncogene. 1993 Oct;8(10):2645-52.
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.
p53肿瘤抑制活性的丧失，无论是通过突变还是通过与人乳头瘤病毒（HPV）E6蛋白相互作用，都被认为是宫颈癌发生的重要机制。我们使用多克隆抗p53抗体和单克隆抗E6抗体研究了这些蛋白在人宫颈癌细胞系中的细胞学分布。通过免疫印迹和免疫竞争分析证实了抗体的特异性。使用传统显微镜技术和三维共聚焦显微镜技术检测了p53和E6的细胞内定位。在HPV - 18或 - 16整合的细胞系HeLa、CaSki和SiHa中，通过免疫荧光在细胞质中观察到病毒癌蛋白E6和内源性肿瘤抑制蛋白p53；在HeLa和CaSki细胞的细胞核中，p53也有微弱的点状染色。在HPV阴性的宫颈癌细胞系C - 33A和HT - 3中，p53发生了突变，p53主要定位于细胞核，与其他细胞系相比，C - 33A细胞中p水平升高。对p53（荧光素）和E6（罗丹明）进行双重荧光染色后，使用共聚焦显微镜对细胞进行了高空间分辨率成像。图像显示p53和E6具有相似的细胞质分布，这意味着这两种蛋白可能以细胞质复合物的形式存在。为了证实这一推测，进行了荧光共振能量转移显微镜检查，这为单个HeLa细胞内p53和E6之间的紧密关联提供了直接证据。本研究结果支持以下理论：p53蛋白在细胞质中与HPV - 16/18 E6蛋白结合，从而阻止p53在细胞核中发挥其肿瘤抑制功能。因此，在宫颈癌中通过形成p53 - E6复合物使野生型p53失活可能是恶性转化的关键步骤。
