|
|
Blood, Vol. 113, Issue 9, 1982-1991, February 26, 2009
Defining the target specificity of ABT-737 and synergistic antitumor activities in combination with histone deacetylase inhibitors
Blood Whitecross et al.
113: 1982
Supplemental materials for: Whitecross et al
Materials and methods
The 3D structures of Bcl-w alone1 or in complex with the BH3 domain of BID2 were downloaded from the MMDB Structure Database3 on the NCBI Web site (MMDB records: 37494 & 22991; PDB records: 1ZY3 & 1MK3) and manipulated using the Cn3D program, version 4.1 (also downloaded from NCBI). To distinguish the bound BH3 domain of BID, the colour scheme of the protein background and helix objects was set to domain by editing the Global Style. The residues of the c-terminal helix a8 (EEARRLREGNWA) and the mouse-specific residues of Bcl-w were highlighted in yellow by selecting them in the sequence viewer. Each highlighted 3D structure was manually rotated in the structure-viewing window and images captured similar orientations. REFERENCES 1. Denisov AY, Madiraju MS, Chen G, et al. Solution structure of human BCL-w: modulation of ligand binding by the C-terminal helix. J Biol Chem. 2003;278:21124–21128.
2. Denisov AY, Chen G, Sprules T, Moldoveanu T, Beauparlant P, Gehring K. Structural model of the BCL-w-BID peptide complex and its interactions with phospholipid micelles. Biochemistry. 2006;45:2250–2256.
3. Chen J, Anderson JB, DeWeese-Scott C, et al. MMDB: Entrez's 3D-structure database. Nucleic Acids Res. 2003;31:474–477.
Files in this Data Supplement:
- Figure S1 (JPG, 101 KB)
-
Whole cell lysates from (A) 4242Eµ-myc and (B) 107Eµ-myc lymphomas transduced with MSCV-IRES-GFP, MSCV-IRES-GFP/Bcl-2, MSCV-IRES-GFP/Bcl-XL, MSCV-IRES-GFP/Flag-Bcl-w, MSCV-IRES-GFP/Flag-Mcl-1, and MSCV-IRES-GFP/Flag-A1 and sorted by flow cytometry for expression of GFP were used for western blot using antibodies against Bcl-2, Bcl-XL, Flag epitope, and, as a loading control, α-tubulin. (C) 4242 Eµ-myc, Eµ-myc/Bcl-2, and Eµ-myc/Mcl-1 cells were treated with increasing concentrations of vorinostat or valproic acid (VPA) for 24 hr. Cells were harvested, fixed, stained with propidium iodide and cell cycle analysis was performed by FACs to assess DNA fragmentation. The percentage of cells with less than 2N DNA content (%subG1) is shown. Results shown are the mean and standard error from at least three separate experiments.
- Figure S2. 107 Eµ-myc/MSCV, Eµ-myc/Bcl-2, Eµ-myc/Bcl-XL, Eµ-myc/Bcl-w, Eµ-myc/Mcl-1, and Eµ-myc/A1 lymphomas were treated for 24 hrs with increasing concentrations of vorinostat (JPG, 53.6 KB)
-
Cell membrane disruption (top panel) and mitochondrial outer membrane permeabilisation (MOMP, bottom panel) were assessed by propidium iodide (PI) and TMRE staining respectively.
- Figure S3. 107 Eµ-myc/MSCV and Eµ-myc/Bcl-2, 226 Eµ-myc/MSCV and Eµ-myc/Bcl-2 cells were treated with increasing concentrations of ABT-737 (A,C) or ABT-737e (B,D) (JPG, 50.5 KB)
-
Cell membrane disruption was assessed by PI staining. Results shown are the mean and standard error from at least three separate experiments.
- Figure S4 (JPG, 76.2 KB)
-
(A) 107 Eµ-myc/Bcl-2, Eµ-myc/Bcl-XL, Eµ-myc/Bcl-w, and Eµ-myc/Mcl-1 lymphomas were treated for 24 hrs with increasing concentrations of ABT-737 or ABT-737e. Cell membrane disruption (left panels) and mitochondrial outer membrane permeabilisation (MOMP, right panels) was assessed by PI and TMRE staining respectively. Results shown are the mean and standard error from at least three separate experiments. (B) 226 Eµ-myc/Bcl-2 and Eµ-myc/Bcl-w lymphomas were treated for 24 hrs with increasing concentrations of ABT-737 or ABT-737e. Cell membrane disruption was assessed by PI staining. Results shown are the mean and standard error from at least three separate experiments.
- Figure S5 (JPG, 60.2 KB)
-
(A) Pairwise alignment of the human (top row) and mouse (mus musculus) Bcl-w amino acid sequences generated using BLAST from the HomoloGene database record 2989 (BCL2L2) for Bcl-w on the NCBI website. Residues of the mouse sequence that differ from human Bcl-w are highlighted in red. (B) Two alternate 3D views of the structure of Bcl-w (shown in pink/purple). On the left is Bcl-w in complex with the BH3 domain of BID (blue) as determined by Denisov et al (2006). On the right is Bcl-w 1 with the c-terminal helix a8 (highlighted in yellow) occupying the groove bound by the BID BH3 domain. The helix a8 residues displaced by the binding of the BID BH3 domain to Bcl-w are also highlighted in the structure on the left. The residues of the mouse sequence that differ from human Bcl-w are marked with * (also highlighted in yellow). Note these are clearly distant from the BH3 binding groove of Bcl-w and the domains that form it, as indicated by the location of the BID BH3 peptide and the helix a8. N and C denote, respectively, the amino and carboxy termini of Bcl-w.
- Figure S6 (JPG, 115 KB)
-
(A) 4242 Eµ-myc/Flag-Bcl-w, Eµ-myc/ Bcl-w, and Eµ-myc/Bcl-2 were treated for 24 hrs with increasing concentrations of ABT-737 or ABT-737e. Cell membrane disruption was assessed by PI staining. Results shown are the mean and standard error from at least three separate experiments. (B) Western blot analysis of 4242 Eµ-myc/Flag-Bcl-w, Eµ-myc/ Bcl-w, Eµ-myc/Bcl-2, and Eµ-myc/MSCV cells using antibodies against Mcl-1, Bcl-w, and tubulin (protein loading control). (C) 4242 Eµ-myc lymphoma cells were transduced with MSCV-IRES-GFP/Flag-hBcl-w (expressing FLAG-tagged human Bcl-w) and sorted by flow cytometry for expression of GFP. Eµ-myc/Bcl-2 and Eµ-myc/hBcl-w cells were treated with increasing concentrations of ABT-737 or ABT-737e (left panels) or vorinostat (right panels) and cells were assessed for uptake of PI, loss of MOMP, and DNA fragmentation (%subG1). Results shown are the mean and standard error from three separate experiments.
- Figure S7 (JPG, 187 KB)
-
(A) 4242 Eµ-myc/MSCV, Eµ-myc/Bcl-2, Eµ-myc/Bcl-XL, Eµ-myc/Bcl-w, Eµ-myc/Mcl-1, and Eµ-myc/A1 lymphomas were treated for 24 hrs with increasing concentrations of VPA (0, 10, 100, 1000 µM) in the presence or absence of ABT-737 or ABT-737e. ABT-737 and ABT-737e was used at a concentration of 0.5 µM for Eµ-myc/Bcl-2, Eµ-myc/Bcl-XL, lymphomas, and at 1.0 µM for Eµ-myc/Bcl-w, Eµ-myc/Mcl-1, and Eµ-myc/A1 cells. Cell membrane disruption (left panels) and mitochondrial outer membrane permeabilisation (MOMP, right panels) was assessed by PI and TMRE staining respectively. (B) 4242 Eµ-myc/Bcl-2, Eµ-myc/Bcl-XL, and Eµ-myc/Bcl-w lymphomas were treated for 24 hrs with increasing concentrations of vorinostat (left panels) or VPA (right panels) in the presence or absence of ABT-737 or ABT-737e. Concentrations used were as in Fig. 4 and Fig. S7A. DNA fragmentation (%subG1) was determined by cell cycle analysis (as in Fig. S1C). Results shown are the mean and standard error from at least three separate experiments.
- Figure S8 (JPG, 84.6 KB)
-
(A) FLR-Eµ-myc/MSCV and FLR-Eµ-myc/Bcl-2 cells (derived from pool #2) were treated for 24 hrs with increasing concentrations of ABT-737. Cell membrane disruption (left panels) and mitochondrial outer membrane permeabilisation (MOMP, right panels) was assessed by PI and TMRE staining respectively. Results shown are the mean and standard error from at least three separate experiments. (B) Western blot confirming over-expression of Bcl-2, Bcl-w, and Mcl-1 (and lack of Bcl-XL expression) in tumours derived from Eµ-myc fetal liver cells. -actin expression serves as a loading control. Tumour cells (except in lane 6) were collected from the enlarged lymph nodes of mice treated with ABT-737 diluent in Fig. 6A. 4242t MSCV-Bcl-XL cells were grown in vitro.
|
|
