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Blood, Vol. 111, Issue 5, 2854-2865, March 1, 2008
Aurora kinase inhibitory VX-680 increases Bax/Bcl-2 ratio and induces apoptosis in Aurora-A-high acute myeloid leukemia
Blood Huang et al.
111: 2854
Supplemental materials for: Huang et al
Files in this Data Supplement:
- Table S1. Clinical characteristics of patients with AML (PDF, 136 KB) -
UPN indicates unique patient number; WBC, white blood cell count; Hb, hemoglobin count; PLT, platelet count; FAB indicates French-American-British classification. WT, wild-type FLT3; ITD, FLT3 internal tandem duplication; TKD, the activation loop of the tyrosine kinase domain. ITD/TKD mutations indicate AML patient had both ITD and TKD mutations. N/A, not assessable. All samples were obtained at diagnosis.
- Figure S1. VX-680 inhibits phosphorylation of both Aur-A (Thr288) and histone H3 (Ser10) (JPG, 61 KB)
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(A)VX-680 inhibits autophosphorylation of Aur-A at Thr288. HL-60 cells were incubated with DMSO or increasing amounts of VX-680 for 24 hrs. Cell lysates were subjected to Western blot analysis with phospho-Aur-A antibody. Note that GAPDH served as a loading control. (B-C) VX-680 inhibits histone H3 phosphorylation at Ser10. Histone H3 phosphorylation at Ser10 was detected by Western blot and immunofluorescence staining with phospho-histone H3-Ser10 specific antibody. Total cell population was visualized by DAPI staining (C, middle). Experiments were repeated three independent times with similar results.
- Figure S2. Suppression of Aur-A, but not Aur-B, preferentially induces apoptosis of AML cells (JPG, 82 KB)
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(A) HL-60 cells were transfected with siRNA duplexes to suppress expression of either Aur-A or Aur-B, as assessed by Western blot analysis. GAPDH represents loading control. (B) Western blot of blasts from patient 37 (M4, Aur-A high expression) transfected with siRNA showing suppression of Aur-A and Aur-B. (C) The apoptosis was assessed by annexin V assays in siRNA transfected cells. Apoptosis of HL-60 cells were observed early at 24 hrs (29.8 ± 3.1%, P<.01) and dramatically increased at 48 hrs (45.1 ± 5.4%, P<.001) after transfection with Aur-A siRNA. In contrast, a significantly less amount of apoptosis (14.1 ± 3.2%) at 24 hrs and (23.5 ± 5%) at 48 hrs was observed after transfection of Aur-B siRNA. The apoptotic degree of control cells was observed as 12.1 ± 1.8% at 24 hrs and 11.8 ± 2% at 48 hrs. (D) Suppression of Aur-A generated a significant amount of apoptosis in AML cells (22.6% at 48 hrs and 54.7% at 72 hrs after RNAi). Aur-B siRNA-transfected AML cells however produced little apoptosis (12.9% at 48 hrs and 19.8% at 72 hrs after RNAi), as assessed by annexin V analysis.
- Figure S3. Expression of Aur-A, but not Aur-B, predicts apoptotic response to VX-680 (JPG, 24.5 KB)
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Dot plot of apoptotic response in individual AML samples to VX-680 (50 nM) was grouped by levels of Aur-B and Aur-A expression. No significant difference of VX-680-induced apoptosis was observed between Aur-B-high and Aur-B-low groups (P >.05).
- Figure S4. VX-680 potentiates VP16-induced cytotoxicity in primary AML cells regardless of the sequence of drug administration (JPG, 20.4 KB)
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Cells from AML blasts were pre-treated with VP16 (10 µM) followed by VX-680 (10nM) (VP16/VX-680) or a combination of both (VP16+VX-680) before staining with annexin V-FITC and PI. The apoptosis were assessed by flow cytometry. No significant difference of apoptosis was observed between sequential (VP16/VX-680) and combination (VP16+VX-680) groups (P >.05). Column, mean; Bars, SD.
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