Miao Ze-Hong, Player Audrey, Shankavaram Uma, Wang Yong-Hong, Zimonjic Drazen B, Lorenzi Philip L, Liao Zhi-Yong, Liu Hong, Shimura Tsutomu, Zhang Hong-Liang, Meng Ling-Hua, Zhang Yong-Wei, Kawasaki Ernest S, Popescu Nicholas C, Aladjem Mirit I, Goldstein David J, Weinstein John N, Pommier Yves
Laboratories of Molecular Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, USA.
Cancer Res. 2007 Sep 15;67(18):8752-61. doi: 10.1158/0008-5472.CAN-06-4554.
The biological functions of nuclear topoisomerase I (Top1) have been difficult to study because knocking out TOP1 is lethal in metazoans. To reveal the functions of human Top1, we have generated stable Top1 small interfering RNA (siRNA) cell lines from colon and breast carcinomas (HCT116-siTop1 and MCF-7-siTop1, respectively). In those clones, Top1 is reduced approximately 5-fold and Top2alpha compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone gamma-H2AX foci associated with replication defects. siTop1 cells also show rDNA and nucleolar alterations and increased nuclear volume. Genome-wide transcription profiling revealed 55 genes with consistent changes in siTop1 cells. Among them, asparagine synthetase (ASNS) expression was reduced in siTop1 cells and in cells with transient Top1 down-regulation. Conversely, Top1 complementation increased ASNS, indicating a causal link between Top1 and ASNS expression. Correspondingly, pharmacologic profiling showed L-asparaginase hypersensitivity in the siTop1 cells. Resistance to camptothecin, indenoisoquinoline, aphidicolin, hydroxyurea, and staurosporine and hypersensitivity to etoposide and actinomycin D show that Top1, in addition to being the target of camptothecins, also regulates DNA replication, rDNA stability, and apoptosis. Overall, our studies show the pleiotropic nature of human Top1 activities. In addition to its classic DNA nicking-closing functions, Top1 plays critical nonclassic roles in genomic stability, gene-specific transcription, and response to various anticancer agents. The reported cell lines and approaches described in this article provide new tools to perform detailed functional analyses related to Top1 function.
细胞核拓扑异构酶I（Top1）的生物学功能一直难以研究，因为敲除TOP1在多细胞动物中是致死的。为了揭示人类Top1的功能，我们分别从结肠癌和乳腺癌中构建了稳定的Top1小干扰RNA（siRNA）细胞系（分别为HCT116-siTop1和MCF-7-siTop1）。在这些克隆中，Top1减少了约5倍，Top2α补偿了Top1的缺陷。siTop1细胞的一个显著特征是基因组不稳定，伴有与复制缺陷相关的染色体畸变和组蛋白γ-H2AX焦点。siTop1细胞还表现出核糖体DNA（rDNA）和核仁改变以及核体积增加。全基因组转录谱分析显示，有55个基因在siTop1细胞中发生了一致的变化。其中，天冬酰胺合成酶（ASNS）在siTop1细胞和瞬时下调Top1的细胞中表达降低。相反，Top1互补增加了ASNS的表达，表明Top1与ASNS表达之间存在因果关系。相应地，药物敏感性分析显示siTop1细胞对L-天冬酰胺酶过敏。对喜树碱、茚并异喹啉、阿非迪霉素、羟基脲和星形孢菌素耐药，对依托泊苷和放线菌素D过敏，这表明Top1除了是喜树碱类药物的靶点外，还调节DNA复制、rDNA稳定性和细胞凋亡。总体而言，我们的研究表明人类Top1活性具有多效性。除了其经典的DNA切口-封闭功能外，Top1在基因组稳定性、基因特异性转录以及对各种抗癌药物的反应中发挥着关键的非经典作用。本文报道的细胞系和方法为进行与Top1功能相关的详细功能分析提供了新工具。
