NF-κB induces abnormal centrosome amplification by upregulation of CDK2 in laryngeal squamous cell cancer

  • Authors: Jian-Li Liu, Hui-Ping Ma, Xiu-Li Lu, Shao-Hua Sun, Xing Guo, Fu-Cai Li
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  • Published online on: Friday, July 15, 2011
  • Pages: 915-924
  • DOI: 10.3892/ijo.2011.1125

Abstract

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.
Journal Cover

October 2011
Volume 39 Issue 4

Print ISSN: 1019-6439
Online ISSN:1791-2423

2013 Impact Factor: 2.773
Ranked #30/202 Oncology
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APA
Liu, J., Ma, H., Lu, X., Sun, S., Guo, X., & Li, F. (2011). NF-κB induces abnormal centrosome amplification by upregulation of CDK2 in laryngeal squamous cell cancer. International Journal of Oncology, 39(4), 915-924.
MLA
Liu, Ma, Lu, Sun, Guo, and Fu-Cai Li. "NF-κB induces abnormal centrosome amplification by upregulation of CDK2 in laryngeal squamous cell cancer." International Journal of Oncology International Journal of Oncology 39.4 (2011): 915-924.
Chicago
Liu, Ma, Lu, Sun, Guo, and Fu-Cai Li. "NF-κB induces abnormal centrosome amplification by upregulation of CDK2 in laryngeal squamous cell cancer." International Journal of Oncology International Journal of Oncology 39 no. 4 (2011): 915-924.