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. 2001 Sep 11;98(19):10833-8.
doi: 10.1073/pnas.191208598. Epub 2001 Sep 4.

The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species

A A Ruefli  1 , M J Ausserlechner, D Bernhard, V R Sutton, K M Tainton, R Kofler, M J Smyth, R W Johnstone
Affiliations

The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species

A A Ruefli et al. Proc Natl Acad Sci U S A. .

Abstract

Many chemotherapeutic agents induce mitochondrial-membrane disruption to initiate apoptosis. However, the upstream events leading to drug-induced mitochondrial perturbation have remained poorly defined. We have used a variety of physiological and pharmacological inhibitors of distinct apoptotic pathways to analyze the manner by which suberoylanilide hydroxamic acid (SAHA), a chemotherapeutic agent and histone deacetylase inhibitor, induces cell death. We demonstrate that SAHA initiates cell death by inducing mitochondria-mediated death pathways characterized by cytochrome c release and the production of reactive oxygen species, and does not require the activation of key caspases such as caspase-8 or -3. We provide evidence that mitochondrial disruption is achieved by means of the cleavage of the BH3-only proapoptotic Bcl-2 family member Bid. SAHA-induced Bid cleavage was not blocked by caspase inhibitors or the overexpression of Bcl-2 but did require the transcriptional regulatory activity of SAHA. These data provide evidence of a mechanism of cell death mediated by transcriptional events that result in the cleavage of Bid, disruption of the mitochondrial membrane, and production of reactive oxygen species to induce cell death.

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Figures

Figure 1
Figure 1
SAHA-induced cell death was not mediated by death receptors. CEM cells expressing sense (CEM-CrmA) or antisense (CEM-CrmA/AS) CrmA cDNA were treated for 24 hr with 0–5 μM SAHA or 12 hr with 100 ng/ml anti-Fas antibody clone CH-11; cell death was assessed by trypan blue. The results shown are representative of three experiments with a number of different CEM-CrmA and CEM-CrmA/AS cell lines. For all cell-death assays, statistical differences (P < 0.05 as determined by the Mann–Whitney U test) between samples are denoted by *.
Figure 2
Figure 2
SAHA-induced cell death is caspase-independent. (A) CEM cells were pretreated with 40 μM ZVAD-fmk or ZFA-fmk for 1 hr and then cultured for 24 hr with 2.5 μM SAHA or 100 ng/ml Vin. Cell death was assessed by trypan blue exclusion. (B) Whole-cell lysates treated with 2.5 μM SAHA in the presence or absence of ZVAD-fmk (V) or ZFA-fmk (F) as above were assessed for PARP cleavage and α-tubulin expression by Western blotting.
Figure 3
Figure 3
SAHA induces equivalent cell death in P-gp+ and P-gp− cells. CEM-P-gp (P-gp+) and CEM (P-gp−) cells were cultured for 24 hr with 0–10 μM SAHA or 100 ng/ml Vin. Cell death was assessed by 51Cr release.
Figure 4
Figure 4
Bcl-2 inhibits SAHA-induced cell death. (A) CEM and CEM-Bcl-2 cells were treated with 0–5 μM SAHA for 24 hr or 100 ng/ml anti-Fas antibody for 12 hr; cell death was assessed by trypan blue exclusion. (B) Western blots of cytosolic extracts from CEM and CEM-Bcl-2 cells treated with or without 2.5 μM SAHA were probed with mAbs to cytochrome c (c) and α-tubulin (t). In some wells, CEM cells were pretreated with 40 μM ZVAD-fmk (lanes 4 and 7) or ZFA-fmk (lanes 5 and 8) for 1 hr and then cultured for 24 hr with 2.5 μM SAHA.
Figure 5
Figure 5
Production of ROS is central to SAHA-mediated cell death. (A) CEM cells were cultured for 24 hr in the presence or absence of 2.5 and 5.0 μM SAHA or 100 ng/ml Vin, labeled with oxidative-sensitive dye C-400, and analyzed by flow cytometry for increases in Fl-1 fluorescence. As a control, cells were labeled with C-400 in the presence of 10 μM H2O2. Values were calculated as the percent mean fluorescence compared with control (untreated cells). (B) Cells were pretreated with CCCP, NAC, or pyrrolidine dithiocarbamate (PDTC) for 1 hr and then cultured for 24 hr with 2.5 μM SAHA; cell death was assessed by trypan blue exclusion.
Figure 6
Figure 6
SAHA induces caspase-independent Bid cleavage in the presence of Bcl-2. (A) Whole-cell lysates from CEM cells cultured for 0–24 hr with 2.5 μM SAHA were assessed for Bid and PARP cleavage by Western blotting. (B) Whole-cell lysates from CEM cells cultured for 24 hr with 2.5 μM SAHA were assessed for Bid cleavage and α-tubulin by Western blot analysis. In some lanes, cells were pretreated for 1 hr with 40 μM ZVAD-fmk (V) or ZFA-fmk (F) and then cultured for 24 hr with 2.5 μM SAHA. As a control for Bid cleavage, whole-cell lysates from Jurkat cells treated with a sublytic dose of perforin in combination with 60 nM GzB (P/G) were run on the same gel. The middle panel is a 30-sec exposure showing the decrease in p28 full-length Bid protein. The bottom panel is a 5-min exposure showing the tBid products p16 and p14. (C) Whole-cell lysates from CEM-Bcl-2 cells treated as above were assessed for Bid cleavage and α-tubulin by Western blotting.
Figure 7
Figure 7
Cooverexpression of Bid reverses Bcl-2's block of SAHA-mediated cell death. FDC-P1 cells (■) and FDC-P1 cells CO-overexpressing Bid (▴), Bcl-2 (●), or Bid and Bcl-2 (□) were cultured for 0–72 hr in the presence of 2.5 μM SAHA (A) or 10 nM STS (B). Cell death was assessed by trypan blue exclusion. These results are representative of six separate experiments.
Figure 8
Figure 8
New gene transcription is necessary for SAHA-mediated cell death. CEM cells were pretreated for 30 min with 10 ng/ml Act D (A) or 500 ng/ml CHX (C) and then cultured for 24 hr in the presence of SAHA. Cell death was assessed by trypan blue exclusion. (B and D) CEM cells were treated as above and whole-cell lysates were assessed for Bid cleavage and α-tubulin protein loading by Western blotting.
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