Figure 5
Figure 5. Aurora kinase activity is important for Aurora-IKK interactions in myeloma cells. (A) HMCLs were cultured with dimethylsulfoxide (vehicle), IKKβ inhibitor PS-1145 (5 μM), or MK-0457 (0.4 μM) for 3 hours and then incubated with TRAIL. After 24 hours, cells were lysed and subjected to western blot analysis to monitor the expression and phosphorylation of IKKβ; anti-actin immunoblot was performed as loading control. (B) The same cell lysates were subjected to immunoprecipitation using mouse monoclonal IKKβ (H-4) or control antibody (IgG) and IB with either IKKβ or Aurora A or Aurora B antibodies. Bands were subjected to densitometric scanning, and the histogram shows average quantification results ± SD of the association Aurora A/IKKβ or Aurora B/IKKβ from 3 blots (*P < .05; **P < .01, vs untreated control cells, Tukey-Kramer test). (C) RPMI 8226 and 8226/R5 cells were cultured with dimethylsulfoxide (vehicle) or PS-1145 at 5 μM for 24 hours. Endogenous pospho-IKKβ, phospho-Aurora A (Thr288), and phospho-Aurora B (Thr232) from whole cell lysates were revealed by western blot analysis. Anti-actin immunoblotting was performed as loading control. The relative amount of phospho-Aurora A (Thr288) and phospho-Aurora B (Thr232) was determined by densitometry and normalized to that of actin. Histograms represent the mean ± SD of the ratio phospho-Aurora A (Thr288)/actin or phospho-Aurora B (Thr232)/actin normalized to the untreated control from 2 blots (*P < .01, Tukey-Kramer test). (D) HMCLs cells were cultured with dimethylsulfoxide (vehicle) or PS-1145 at 5 μM for 3 hours and then incubated with TRAIL (2.4 ng/mL in RPMI 8226, 19.2 ng/mL in RPMI 8226/R5, and 300 ng/mL in JJN3) and after 24 hours, the percentage of cell death was determined by the annexin-V method. Values are mean ± SD of 3 independent experiments. (E) Model of TRAIL action; TRAIL induces prosurvival signal in MM cells by activating both Aurora A and B kinases, leading to increased Aurora/IKK binding, IKK phosphorylation/activation, NF-κB activation, and induction of the antiapoptotic NF-κB target genes A1/Bfl-1, Mcl-1, and IAPs. Aurora kinase inhibitors disable TRAIL-directed survival pathways, because they abrogate TRAIL-induced Aurora-IKK kinase interactions and NF-κB activation.

Aurora kinase activity is important for Aurora-IKK interactions in myeloma cells. (A) HMCLs were cultured with dimethylsulfoxide (vehicle), IKKβ inhibitor PS-1145 (5 μM), or MK-0457 (0.4 μM) for 3 hours and then incubated with TRAIL. After 24 hours, cells were lysed and subjected to western blot analysis to monitor the expression and phosphorylation of IKKβ; anti-actin immunoblot was performed as loading control. (B) The same cell lysates were subjected to immunoprecipitation using mouse monoclonal IKKβ (H-4) or control antibody (IgG) and IB with either IKKβ or Aurora A or Aurora B antibodies. Bands were subjected to densitometric scanning, and the histogram shows average quantification results ± SD of the association Aurora A/IKKβ or Aurora B/IKKβ from 3 blots (*P < .05; **P < .01, vs untreated control cells, Tukey-Kramer test). (C) RPMI 8226 and 8226/R5 cells were cultured with dimethylsulfoxide (vehicle) or PS-1145 at 5 μM for 24 hours. Endogenous pospho-IKKβ, phospho-Aurora A (Thr288), and phospho-Aurora B (Thr232) from whole cell lysates were revealed by western blot analysis. Anti-actin immunoblotting was performed as loading control. The relative amount of phospho-Aurora A (Thr288) and phospho-Aurora B (Thr232) was determined by densitometry and normalized to that of actin. Histograms represent the mean ± SD of the ratio phospho-Aurora A (Thr288)/actin or phospho-Aurora B (Thr232)/actin normalized to the untreated control from 2 blots (*P < .01, Tukey-Kramer test). (D) HMCLs cells were cultured with dimethylsulfoxide (vehicle) or PS-1145 at 5 μM for 3 hours and then incubated with TRAIL (2.4 ng/mL in RPMI 8226, 19.2 ng/mL in RPMI 8226/R5, and 300 ng/mL in JJN3) and after 24 hours, the percentage of cell death was determined by the annexin-V method. Values are mean ± SD of 3 independent experiments. (E) Model of TRAIL action; TRAIL induces prosurvival signal in MM cells by activating both Aurora A and B kinases, leading to increased Aurora/IKK binding, IKK phosphorylation/activation, NF-κB activation, and induction of the antiapoptotic NF-κB target genes A1/Bfl-1, Mcl-1, and IAPs. Aurora kinase inhibitors disable TRAIL-directed survival pathways, because they abrogate TRAIL-induced Aurora-IKK kinase interactions and NF-κB activation.

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