Department of Medical Genetics, China Medical University, Shenyang 110001, PR China.
Int J Oncol. 2011 Oct;39(4):915-24. doi: 10.3892/ijo.2011.1125. Epub 2011 Jul 15.
Centrosome amplification can drive chromosomal instability (CIN) which is a major source of tumor initiation. The present study aimed to investigate the impact of nuclear factor kappa B (NF-κB) on centrosome amplification of Hep-2 cells. Immunofluorescence was performed to display centrosomes. BAY11-7082 was used as an inhibitor of NF-κB to assess the inhibition of centrosome amplification, and cyclin-dependent kinase 2 (CDK2), ensuring cell cycle cycle coordination with centrosome cycle was detected by Western blotting. Furthermore, a 1556-bp fragment of the CDK2 promoter was analyzed using the TRANSFAC-TESS software. Luciferase assay, including a series of truncated CDK2 promoters and site mutations, was carried out to determine NF-κB binding sites in the CDK2 promoter. Electrophoresis mobility shift and chromatin immunoprecipitation assays were applied to confirm whether NF-κB indeed binds to the 5'-promoter region of the CDK2 gene. To reveal the clinical significance of CDK2 expression in laryngeal squamous cell cancer, mRNA and protein levels were assessed by RT-PCR and Western blotting, respectively. We found that the transcription factor NF-κB plays a role in centrosome amplification in Hep-2 cells. Centrosome amplification is reduced by inhibition of the NF-κB pathway. Moreover, expression of the p65 subunit of NF-κB is sufficient to promote centrosome amplification and increase in CDK2 protein levels. We further identified a functional NF-κB binding site located in the CDK2 promoter. Single mutation of the NF-κB site III (construct mutIII) however resulted in 76±5% (p<0.01) luciferase activity reduction. Electromobility shift assays and chromatin immunoprecipitaton results suggest that NF-κB indeed binds to this responsive element associating with CDK2 expression and centrosome amplification. RT-PCR and Western blotting results revealed that both mRNA and protein levels of CDK2 were significantly higher in tumor tissues than those in paired adjacent normal laryngeal tissues.
中心体扩增可导致染色体不稳定（CIN），这是肿瘤发生的主要来源。本研究旨在探讨核因子 kappa B（NF-κB）对 Hep-2 细胞中心体扩增的影响。免疫荧光法显示中心体。BAY11-7082 被用作 NF-κB 的抑制剂，以评估对中心体扩增的抑制作用，并通过 Western blot 检测细胞周期与中心体周期协调的细胞周期蛋白依赖性激酶 2（CDK2）。此外，使用 TRANSFAC-TESS 软件分析了 CDK2 启动子的 1556bp 片段。进行了荧光素酶测定，包括一系列截短的 CDK2 启动子和位点突变，以确定 CDK2 启动子中 NF-κB 的结合位点。电泳迁移率变动和染色质免疫沉淀测定用于证实 NF-κB 确实与 CDK2 基因的 5'-启动子区域结合。为了揭示 CDK2 表达在喉鳞状细胞癌中的临床意义，通过 RT-PCR 和 Western blot 分别评估了 mRNA 和蛋白水平。我们发现转录因子 NF-κB 在 Hep-2 细胞中发挥作用中心体扩增。NF-κB 通路的抑制可减少中心体扩增。此外，NF-κB p65 亚基的表达足以促进中心体扩增并增加 CDK2 蛋白水平。我们进一步确定了位于 CDK2 启动子中的功能性 NF-κB 结合位点。然而，NF-κB 位点 III（构建 mutIII）的单突变导致荧光素酶活性降低 76±5%（p<0.01）。电泳迁移率变动测定和染色质免疫沉淀结果表明，NF-κB 确实与该反应元件结合，与 CDK2 表达和中心体扩增相关。RT-PCR 和 Western blot 结果表明，肿瘤组织中 CDK2 的 mRNA 和蛋白水平均明显高于配对的相邻正常喉组织。
