Dall Keltie L, Scarpini Cinzia G, Roberts Ian, Winder David M, Stanley Margaret A, Muralidhar Balaji, Herdman M Trent, Pett Mark R, Coleman Nicholas
Medical Research Council-Cancer Cell Unit, Hutchison/Medical Research Council Research Center, University of Cambridge, Cambridge, United Kingdom.
Cancer Res. 2008 Oct 15;68(20):8249-59. doi: 10.1158/0008-5472.CAN-08-1741.
As the high-risk human papillomavirus (HPV) integrants seen in anogenital carcinomas represent the end-point of a clonal selection process, we used the W12 model to study the naturally occurring integration events that exist in HPV16-infected cervical keratinocytes before integrant selection. We performed limiting dilution cloning to identify integrants present in cells that also maintain episomes. Such integrants arise in a natural context and exist in a noncompetitive environment, as they are transcriptionally repressed by episome-derived E2. We found that integration can occur at any time during episome maintenance, providing biological support for epidemiologic observations that persistent HPV infection is a major risk factor in cervical carcinogenesis. Of 24 different integration sites isolated from a single nonclonal population of W12, 12 (50%) occurred within chromosome bands containing a common fragile site (CFS), similar to observations for selected integrants in vivo. This suggests that such regions represent relatively accessible sites for insertion of foreign DNA, rather than conferring a selective advantage when disrupted. Interestingly, however, integrants and CFSs did not accurately colocalize. We further observed that local DNA rearrangements occur frequently and rapidly after the integration event. The majority of integrants were in chromosome bands containing a cancer-associated coding gene or microRNA, indicating that integration occurs commonly in these regions, regardless of selective pressure. The cancer-associated genes were generally a considerable distance from the integration site, and there was no evidence for altered expression of nine strong candidate genes. These latter observations do not support an important role for HPV16 integration in causing insertional mutagenesis.
由于在肛门生殖器癌中发现的高危人乳头瘤病毒（HPV）整合体代表了克隆选择过程的终点，我们使用W12模型来研究在整合体选择之前HPV16感染的宫颈角质形成细胞中存在的自然发生的整合事件。我们进行了有限稀释克隆，以鉴定存在于同时维持游离型病毒基因组的细胞中的整合体。这些整合体在自然环境中产生，并存在于非竞争性环境中，因为它们受到游离型病毒基因组衍生的E2的转录抑制。我们发现整合可以在游离型病毒基因组维持期间的任何时间发生，这为流行病学观察提供了生物学支持，即持续的HPV感染是宫颈癌发生的主要危险因素。从单个非克隆的W12群体中分离出的24个不同的整合位点中，有12个（50%）发生在含有常见脆性位点（CFS）的染色体带内，这与体内选定整合体的观察结果相似。这表明这些区域代表了外源DNA插入的相对易接近位点，而不是在被破坏时赋予选择优势。然而，有趣的是，整合体和CFS并没有准确地共定位。我们进一步观察到，整合事件后局部DNA重排频繁且迅速发生。大多数整合体位于含有癌症相关编码基因或微小RNA的染色体带内，这表明整合在这些区域普遍发生，而与选择压力无关。癌症相关基因通常与整合位点有相当的距离，并且没有证据表明9个强候选基因的表达发生改变。这些后一种观察结果不支持HPV16整合在导致插入诱变中起重要作用。
