title: NF-κB induces abnormal centrosome amplification by upregulation of CDK2 in laryngeal squamous cell cancer.
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.
authors.affiliation: Department of Medical Genetics, China Medical University, Shenyang 110001, PR China.
authors.forename: Jian-Li
authors.initials: JL
authors.lastname: Liu
authors.forename: Hui-Ping
authors.initials: HP
authors.lastname: Ma
authors.forename: Xiu-Li
authors.initials: XL
authors.lastname: Lu
authors.forename: Shao-Hua
authors.initials: SH
authors.lastname: Sun
authors.forename: Xing
authors.initials: X
authors.lastname: Guo
authors.forename: Fu-Cai
authors.initials: FC
authors.lastname: Li
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D018385:Centrosome / Q000187:drug effects / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D005784:Gene Amplification; D057809:HEK293 Cells; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D018307:Neoplasms, Squamous Cell / Q000235:genetics* / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D021122:Protein Subunits; D013450:Sulfones / Q000494:pharmacology; D015533:Transcriptional Activation; D014162:Transfection; D015854:Up-Regulation
chemical_list: C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D009570:Nitriles; D021122:Protein Subunits; D013450:Sulfones; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2