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Molecular Cancer Therapeutics
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Enhanced Fas-associated death domain recruitment by histone deacetylase inhibitors is critical for the sensitization of chronic lymphocytic leukemia cells to TRAIL-induced apoptosis

Satoshi Inoue, Nick Harper, Renata Walewska, Martin J.S. Dyer and Gerald M. Cohen
Satoshi Inoue
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Nick Harper
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Renata Walewska
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Martin J.S. Dyer
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Gerald M. Cohen
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DOI: 10.1158/1535-7163.MCT-09-0451 Published November 2009
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    Figure 1.

    Depsipeptide does not enhance TRAIL-R1 aggregation and the redistribution of DISC components to lipid rafts. CLL cells were cultured for 16 h either alone (Con) or with depsipeptide (Dep; 10 nmol/L) and then (A) exposed to TRAIL (500 ng/mL) for the indicated times. Aggregation of TRAIL-R1 was analyzed by Western blotting under nonreducing conditions or (B) exposed to b-TRAIL (500 ng/mL) for 0.5 h. Lipid rafts were isolated and fractions (1 mL) were separated by discontinuous sucrose gradients and analyzed by Western blotting. C, CLL cells were treated as in A and then labeled with cleavable biotin as described in Materials and Methods. Cells were then exposed to TRAIL (500 ng/mL) for the indicated times, and following removal of cell surface biotin, internalization of TRAIL-R1 was detected by Western blotting.

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    Figure 2.

    Recruitment of FADD is critical for HDACi-mediated sensitization to TRAIL-induced apoptosis. A, CLL cells were incubated for 16 h either alone or in the presence of depsipeptide (10 nmol/L). Cells were exposed to PMA (20 ng/mL) for 30 min and/or bisindolylmaleimide I (Bis I; 1 μmol/L) for 60 min before TRAIL (500 ng/mL) for a further 4 h when cells were assessed for apoptosis by phosphatidylserine (PS) externalization. Columns, mean (n = 9); bars, SD. B, CLL cells were treated as in A, except that cells were also exposed to Go6976 60 min before TRAIL exposure, and apoptosis was assessed as in A. Cells were also exposed to b-TRAIL (500 ng/mL) for 15 min and the DISC was isolated as described in Materials and Methods and examined by Western blotting.

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    Figure 3.

    Pretreatment with depsipeptide results in an increase in phospho-FADD in the DISC. CLL cells were exposed either to depsipeptide (10 nmol/L) for 16 h or PMA (20 ng/mL) for 30 min or a combination of both followed by b-TRAIL (500 ng/mL) at 37°C for 15 min. The TRAIL DISC was isolated and two-dimensional gel electrophoresis from either isolated DISC or lysate samples was carried out. A, TRAIL-R1 was detected in the isolated DISC. B, FADD was detected in the DISC and lysate. C, control CLL cells or cells exposed to depsipeptide (10 nmol/L) for 16 h were then treated for the indicated times with b-TRAIL (500 ng/mL) at 25°C to slow down DISC formation. The isolated DISC samples and the lysate were subjected to two-dimensional gel electrophoresis and FADD was detected by Western blotting. D, CLL cells were exposed to depsipeptide (10 nmol/L) for 16 h followed by b-TRAIL for 15 min. The TRAIL DISC and the lysate samples were incubated alone or in the presence of λ phosphatase at 30°C for 30 min and analyzed for the presence of FADD or phospho-FADD (S194).

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    Figure 4.

    PKCβ, lymphocyte-specific protease-1 (LSP1), Lyn, Syk, and SHP-1 are detected in the isolated DISC. CLL cells were exposed to depsipeptide (10 nmol/L) for 16 h followed by ST-TRAIL (500 ng/mL) at 37°C and the TRAIL DISC was isolated. A, protein samples of both lysate and the DISC from two patients were analyzed by Western blotting. B, protein samples of the isolated DISC from control or depsipeptide-exposed cells at the indicated times were analyzed. C, protein samples of the lysate and the receptor pulled down from unstimulated (u/s) cells as well as the isolated DISC from control or depsipeptide-exposed cells were analyzed.

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    Figure 5.

    BCR signaling and tyrosine kinase activity are not important for TRAIL sensitivity. A, CLL cells were incubated for 16 h either alone or with depsipeptide (10 nmol/L). Cells were then exposed to anti-IgM antibody for 60 min before TRAIL (500 ng/mL) for a further 4 h and apoptosis was assessed. Columns, mean (n = 8); bars, SD. B, CLL cells were exposed as indicated either for 16 h to depsipeptide (lanes 3 and 4) or for 2 h to either PP2 (lanes 5-10), SU6656 (SU; lanes 11-16), or piceatannol (Pic; lanes 17-20). Cells were then exposed to TRAIL (TR; 500 ng/mL) for 4 h and apoptosis was assessed. Columns, mean (n = 5); bars, SD. C, CLL cells were exposed for 16 h either alone (lane 1) or to HDACi [trichostatin A (TSA); 0.25 μmol/L; lane 2], depsipeptide (10 nmol/L; lane 3), LBH589 (LBH; 10 nmol/L; lane 4), or valproate (VPA; 2.5 mmol/L; lane 5). Cells were also exposed to bisindolylmaleimide I (1 μmol/L; lanes 6 and 8) for 1 h, PMA (20 ng/mL; lanes 7 and 8) for 30 min, or PP2 (1-10 μmol/L; lanes 9-11) for 2 h. Protein samples were prepared before TRAIL exposure and analyzed by Western blotting. Cells were also further incubated in the presence or absence of TRAIL (500 ng/mL) for 4 h and apoptosis was assessed.

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Molecular Cancer Therapeutics: 8 (11)
November 2009
Volume 8, Issue 11
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Enhanced Fas-associated death domain recruitment by histone deacetylase inhibitors is critical for the sensitization of chronic lymphocytic leukemia cells to TRAIL-induced apoptosis
Satoshi Inoue, Nick Harper, Renata Walewska, Martin J.S. Dyer and Gerald M. Cohen
Mol Cancer Ther November 1 2009 (8) (11) 3088-3097; DOI: 10.1158/1535-7163.MCT-09-0451

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Enhanced Fas-associated death domain recruitment by histone deacetylase inhibitors is critical for the sensitization of chronic lymphocytic leukemia cells to TRAIL-induced apoptosis
Satoshi Inoue, Nick Harper, Renata Walewska, Martin J.S. Dyer and Gerald M. Cohen
Mol Cancer Ther November 1 2009 (8) (11) 3088-3097; DOI: 10.1158/1535-7163.MCT-09-0451
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