Pett Mark R, Alazawi William O F, Roberts Ian, Dowen Sally, Smith David I, Stanley Margaret A, Coleman Nicholas
Medical Research Council Cancer Cell Unit, MRC/Hutchison Research Centre, University of Cambridge, Hills Road, Cambridge CB2 2XZ, United Kingdom.
Cancer Res. 2004 Feb 15;64(4):1359-68. doi: 10.1158/0008-5472.can-03-3214.
Whereas two key steps in cervical carcinogenesis are integration of high-risk human papillomavirus (HR-HPV) and acquisition of an unstable host genome, the temporal association between these events is poorly understood. Chromosomal instability is induced when HR-HPV E7 oncoprotein is overexpressed from heterologous promoters in vitro. However, it is not known whether such events occur at the "physiologically" elevated levels of E7 produced by deregulation of the homologous HR-HPV promoter after integration. Indeed, an alternative possibility is that integration in vivo is favored in an already unstable host genome. We have addressed these issues using the unique human papillomavirus (HPV) 16-containing cervical keratinocyte cell line W12, which was derived from a low-grade squamous intraepithelial lesion and thus acquired HPV16 by "natural" infection. Whereas W12 at low passage contains HPV16 episomes only, long-term culture results in the emergence of cells containing integrated HPV16 only. We show that integration of HPV16 in W12 is associated with 3' deletion of the E2 transcriptional repressor, resulting in deregulation of the homologous promoter of the integrant and an increase in E7 protein levels. We further demonstrate that high-level chromosomal instability develops in W12 only after integration and that the forms of instability observed correlate with the physical state of HPV16 DNA and the level of E7 protein. Whereas intermediate E7 levels are associated with numerical chromosomal abnormalities, maximal levels are associated with both numerical and structural aberrations. HR-HPV integration is likely to be a critical event in cervical carcinogenesis, preceding the development of chromosomal abnormalities that drive malignant progression.
鉴于宫颈癌发生过程中的两个关键步骤是高危型人乳头瘤病毒（HR-HPV）的整合以及宿主基因组获得不稳定性，这些事件之间的时间关联却鲜为人知。在体外，当HR-HPV E7癌蛋白从异源启动子过度表达时会诱导染色体不稳定。然而，尚不清楚在整合后同源HR-HPV启动子失调所产生的E7“生理”升高水平时是否会发生此类事件。实际上，另一种可能性是体内整合在已经不稳定的宿主基因组中更易发生。我们使用独特的含人乳头瘤病毒（HPV）16的宫颈角质形成细胞系W12来解决这些问题，该细胞系源自低度鳞状上皮内病变，因此通过“自然”感染获得HPV16。低传代的W12仅含有HPV16游离基因，而长期培养则导致仅含有整合型HPV16的细胞出现。我们表明，W12中HPV16的整合与E2转录抑制因子的3'缺失相关，导致整合体同源启动子失调以及E7蛋白水平增加。我们进一步证明，W12仅在整合后才出现高水平的染色体不稳定，并且观察到的不稳定形式与HPV16 DNA的物理状态和E7蛋白水平相关。中等E7水平与染色体数目异常相关，而最高水平则与数目和结构畸变均相关。HR-HPV整合很可能是宫颈癌发生中的关键事件，先于驱动恶性进展的染色体异常的发生。
