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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Inhibition of Histone Deacetylase Class I but not Class II Is Critical for the Sensitization of Leukemic Cells to Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis

Satoshi Inoue, Antonello Mai, Martin J.S. Dyer and Gerald M. Cohen
DOI: 10.1158/0008-5472.CAN-05-4563 Published July 2006

Abstract

From work done largely on derived cell lines, it has been suggested that tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) might be a therapeutic target for many forms of malignancy. However, use of primary tumor cells, including chronic lymphocytic leukemic (CLL) cells, has shown inherent resistance to TRAIL. Although the molecular basis for this resistance remains unknown, treatment with histone deacetylase inhibitors (HDACi) often sensitizes resistant cells to TRAIL-induced apoptosis. We used structurally diverse HDACi to ascertain which HDAC needs to be inhibited for the sensitization. Inhibition of HDAC class I but not class II is required for sensitization to TRAIL-induced apoptosis of CLL cells and various cell lines. Using different HDACi together with small interfering RNA for HDAC1, HDAC2, HDAC3, and HDAC6, we report that inhibition of HDAC1 and HDAC2 but not HDAC3, HDAC6, and HDAC8 are primarily responsible for sensitization to TRAIL-induced apoptosis. Based on these data and our previous studies, we propose that a clinical trial in CLL is warranted using a combination of a selective HDACi that inhibits HDAC1 and/or HDAC2 together with a form of TRAIL that signals through TRAIL receptor 1. (Cancer Res 2006; 66(13): 6785-92)

  • Histone deacetylase inhibitors
  • CLL
  • apoptosis
  • sensitization to TRAIL

Introduction

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis selectively in many cancer cells but not in most normal cells and has been proposed as a valuable antitumor agent ( 1). TRAIL interacts with two membrane-bound death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5/TRICK2), and two putative membrane-bound decoy receptors, TRAIL-R3 and TRAIL-R4, and a soluble receptor, osteoprotegrin (reviewed in ref. 1). Triggering of the receptor results in recruitment of the adaptor molecule FADD/MORT1, which, in turn, recruits and activates caspase-8 in the death-inducing signaling complex (DISC; refs. 2– 4). Active caspase-8 then activates other caspases either directly or indirectly through an amplification pathway involving mitochondria and caspase-8-mediated cleavage of Bid, which seems particularly important in TRAIL-induced apoptosis ( 5).

Resistance to TRAIL is an important therapeutic problem that may be circumvented by combination treatments that act by various mechanisms, including a decrease in c-FLIP levels or restoration of caspase-8 expression ( 6). As most agents used in such combinations are inherently toxic, it is imperative to find nontoxic agents, such as histone deacetylase (HDAC) inhibitors (HDACi), which have recently entered clinical trials and exert their antitumor effects by inducing growth arrest, differentiation, and apoptosis ( 7– 10). Posttranslational modifications of histones, particularly acetylation, are important in regulating gene expression ( 7, 11). The acetylation status of core histones is regulated by the balance between histone acetyltransferases and HDACs ( 7, 11, 12). Changes in the structure or expression of histone acetyltransferases and HDACs are common features of many cancers ( 7, 11). There are three classes of mammalian HDAC enzymes, class I comprising HDAC1, HDAC2, HDAC3, and HDAC8; class II HDACs comprising HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10; and a third class, which are NAD-dependent Sir2 deacetylases ( 9– 11). HDACi that inhibit class I and II HDACs do not inhibit Sir2 deacetylases ( 13). Relatively little is known about the recently identified class III HDACs. HDACi alone induce apoptosis primarily by the intrinsic pathway with caspase-9 as the apical caspase ( 14, 15). HDACi can also potentiate death receptor–induced apoptosis ( 16– 18) by various mechanism(s), including increased expression of death receptors and their ligands, decreases in c-FLIP, or redistribution of death receptors to lipid rafts ( 19– 22).

Cells from patients with chronic lymphocytic leukemia (CLL), which are inherently resistant to TRAIL ( 23), are sensitized by prior treatment with HDACi by facilitating increased formation of the TRAIL DISC ( 24). Similarly, another HDACi, LAQ824, enhanced TRAIL-induced apoptosis by increasing DISC activity ( 25). HDACi-mediated sensitization of CLL cells occurs almost exclusively through TRAIL-R1, not TRAIL-R2 ( 26, 27). These data highlighted the possibility of treating CLL with a combination of an HDACi and a form of TRAIL that signals through TRAIL-R1, such as HGS-ETR1, a TRAIL-R1 agonistic antibody, or a mutant form of TRAIL that signals specifically through TRAIL-R1 ( 27). The present study was undertaken to determine which HDAC should be inhibited to sensitize to TRAIL-induced apoptosis and also which HDACi may be optimal to use with TRAIL in combination therapy. We now show that class I but not class II HDACs are involved in sensitization to TRAIL.

Materials and Methods

Lymphocyte purification, cell lines, and culture. CLL cells, obtained with patient consent and local ethical committee approval, were purified and cultured in RPMI 1640 (4 × 106/mL) as described ( 24, 26). Jurkat T cells (clone E6-1), U937 cells, and K562 cells were cultured as previously described ( 24, 26). DU145 cells, from Dr. E. Tulchinsky (University of Leicester, Leicester, United Kingdom), were cultured in RPMI 1640.

Materials. Medium and serum were from Life Technologies, Inc. (Paisley, United Kingdom). Human recombinant TRAIL was prepared as previously described ( 28). Rabbit anti-HDAC1, anti-HDAC3, anti-HDAC4, anti-HDAC5, anti-HDAC6, and anti-HDAC7 antibodies were from Cell Signalling Technology (Beverly, MA); rabbit anti-HDAC2 antibody was from Zymed Laboratories, Inc. (South San Francisco, CA); and anti-HDAC8 monoclonal antibody (mAb; clone HDAC8-48) and antiacetylated tubulin mAb (clone 6-11B-1) were from Sigma (Poole, United Kingdom). The antibody to poly(ADP-ribose)polymerase (PARP; clone C2-10) was from Alexis Corp. (San Diego, CA). Rabbit anti-acetylated-H3 and anti-acetylated-H4 antibodies were from Upstate Biotechnology (Lake Placid, NY). Depsipeptide and cyclic hydroxamic acid–containing peptide 31 (CHAP31; ref. 29) were kindly provided by Dr. E. Sausville (National Cancer Institute, Bethesda, MD) and Dr. Yoshida (RIKEN Institute, Wako, Saitama, Japan), respectively. MS-275 and valproate were from Calbiochem (La Jolla, CA). MC1575 and MC1568, both (aryloxo-propenyl)pyrroyl hydroxamates HDAC class II inhibitors, were synthesized as described (refs. 30, 31; these compounds are called 2c and 2f, respectively, in ref. 31). SB-379278A, an HDAC8 inhibitor, and SB556629 (Scriptaid) were from Dr. Hu (Glaxo SmithKline, King of Prussia, PA; ref. 32). Other reagents, including anti-caspase-3 and anti-caspase-8 antibodies, were from previously described sources ( 24). HGS-ETR1 and HGS-ETR2, agonistic antibodies to TRAIL-R1 and TRAIL-R2, respectively, were from Human Genome Sciences (Rockville, MD).

Quantification of apoptosis and Western blot analysis. Apoptosis was quantified by phosphatidylserine externalization in the presence of propidium iodide ( 24). Samples for Western blot analysis were prepared, and caspases and cleaved PARP were detected as described ( 23, 24, 33). Extraction of histones for the detection of acetylated histone H3 and H4 were prepared as described ( 24). Nuclei, for detection of HDAC8 by Western blot analysis, were prepared by standard methods.

Small RNA interference of HDAC1, HDAC2, HDAC3, and HDAC6. DU145 cells were seeded at 0.5 × 105 per well in 12-well plates and incubated for 24 hours. Cells were transfected with predesigned small interfering RNA (siRNA) oligonucleotides (Ambion, Austin, TX) at a final concentration of 20 nmol/L (HDAC1, HDAC2, and HDAC3) or 30 nmol/L (HDAC6) using Effectene (Qiagen, Crawley, West Sussex, United Kingdom). After 12 hours, medium was changed to fresh prewarmed RPMI medium, incubated for a further 24 hours, and samples were prepared for Western blotting ( 23, 24). The sequences used for RNA interference (RNAi) were as follows; HDAC1a sense, 5′-(GGAGAUGACCAAGUACCACtt)-3′; HDAC1a antisense, 5′-(GUGGUACUUGGUCAUCUCCtc)-3′; HDAC1b sense, 5′-(GGACUGUCCAGUAUUCGAUtt)-3′; HDAC1b antisense, 5′-(AUCGAAUACUGGACAGUCCtc)-3′; HDAC1c sense, 5′-(GGCAAGUAUUAUGCUGUUAtt)-3′; HDAC1c antisense, 5′-(UAACAGCAUAAUACUUGCCtt)-3′; HDAC2a sense, 5′-(GGAUUACAUCAUGCUAAGAtt)-3′; HDAC2a antisense, 5′-(UCUUAGCAUGAUGUAAUCCtc)-3′; HDAC2b sense, 5′-(GGAUUCUGUUACGUUAAUGtt)-3′; HDAC2b antisense, 5′-(CAUUAACGUAACAGAAUCCtg)-3′; HDAC2c sense, 5′-(GGAAGAAGAUAAAUCCAAGtt)-3′; HDAC2c antisense, 5′-(CUUGGAUUUAUCUUCUUCCtt)-3′; HDAC3a sense, 5′-(GGACUACAUUGACUUCCUGtt)-3′; HDAC3a antisense, 5′-(CAGGAAGUCAAUGUAGUCCtc)-3′; HDAC3b sense, 5′-(GGCUUCACCAAGAGUCUUAtt)-3′; HDAC3b antisense, 5′-(UAAGACUCUUGGUGAAGCCtt)-3′; HDAC3c sense, 5′-(GGAAUUUCUUUUCACCUCUtt)-3′; HDAC3c antisense, 5′-(AGAGGUGAAAAGAAAUUCCtt)-3′; HDAC6a sense, 5′-(GCUCGGCCAAGCAAUGGAAtt)-3′; HDAC6a antisense, 5′-(UUCCAUUGCUUGGCCGAGCtt)-3′; HDAC6b sense, 5′-(GCAGUUAAAUGAAUUCCAUtt)-3′; HDAC6b antisense, 5′-(AUGGAAUUCAUUUAACUGCtc)-3′; HDAC6c sense, 5′-(GCCUAGAAUAUAUUGAUCUtt)-3′; HDAC6c antisense, 5′-(AGAUCAAUAUAUUCUAGGCtg)-3′.

HDAC activity measurements following immunoprecipitation of HDAC1, HDAC2, and HDAC3. Cells (7 × 106) were lysed in buffer [20 mmol/L Tris-HCl (pH 8), 0.15 mol/L NaCl, 10% glycerol, and 0.5% NP40] for 20 minutes on ice. After centrifugation (12,000 × g for 10 minutes), the supernatant fraction (cell lysate) was incubated with 2 μg rabbit IgG, anti-HDAC1, or anti-HDAC2 (Cyclex, Nagano, Japan) or 5 μg HDAC3 (Bethyl Labs, Montgomery, TX) overnight at 4°C followed by incubation with 50 μL of protein Sepharose A beads (Amersham, Little Chalfont, Bucks, United Kingdom) for 3 hours at 4°C. Beads were washed thrice in PBS and resuspended in HDAC assay buffer [50 μL; 20 mmol/L Tris-HCl (pH 8), 125 mmol/L NaCl, and 1% glycerol]. HDAC1 and HDAC2 activities were measured using 5 μL of the sample according to the procedure in the Cyclex HDAC Assay kit (Cyclex). HDAC3 activity was measured according to procedure in the HDAC fluorimetric assay/drug discovery kit (AK-500, Biomol International LP, Plymouth Meeting, PA).

Statistical analysis. A one-tailed paired t test was used because of the explicit expectation that HDACi would sensitize CLL cells to TRAIL-induced apoptosis based on our previous studies ( 24, 26). All results with individual HDACi treatments were compared with their corresponding controls without the HDACi. When multiple comparisons were made within an experiment, Bonferroni correction was applied.

Results

Expression of HDACs in CLL cells and various cell lines. Previously, we had shown that some HDACi potentiated TRAIL-induced apoptosis in various cell types, including Jurkat, U937, K562, and freshly isolated CLL cells ( 24). To ascertain which specific HDAC was inhibited to sensitize to TRAIL, we used a panel of antibodies to determine the expression of different HDACs ( Fig. 1 ). All the cells expressed both class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8) and some class II HDACs (HDAC6 and HDAC7; Fig. 1). As no detectable HDAC4 and HDAC5 was found in these cells, inhibition of these HDACs is not required for potentiation of TRAIL-induced apoptosis.

Figure 1.
Figure 1.

Expression of HDAC1-8 in different cells. Jurkat, U937, K562, and primary CLL cells were examined by Western blotting for the expression of HDAC1-8 as described in Materials and Methods. Equal amounts of protein, determined by the Bradford assay, were loaded onto the gels and confirmed by staining the membranes with Ponceau S.

Inhibition of class I but not class II HDACs is important for TRAIL sensitization. To determine if it was necessary to inhibit any specific HDAC to potentiate TRAIL, we used structurally diverse HDACi with differing specificities. As the HDAC expression pattern was similar in the cell lines and CLL cells, we initially used Jurkat and K562 cells to ascertain which HDACs may be important for sensitization because of the ease of using cell lines and also to overcome some of the variation in using cells from different patients. Jurkat and K562 cells were exposed to trichostatin A (TSA), a broad-spectrum HDACi, which inhibits both class I and II HDACs ( 34). TSA caused a marked hyperacetylation of histone H3, H4, and tubulin ( Fig. 2A and B, lane 2 ), confirming it inhibited both class I and II HDACs. Accumulation of acetylated tubulin results from inhibition of the major cellular tubulin deacetylase, HDAC6, a class II HDAC ( 34, 35). TSA also potentiated TRAIL-induced apoptosis in both Jurkat and K562 cells, as assessed by phosphatidylserine externalization, processing of caspase-3 to its catalytically active large subunits (p19/p17) and cleavage of PARP to yield its characteristic product of 85 kDa ( Fig. 2C and D, lanes 3 and 4), suggesting that inhibition of HDAC class I and/or class II may be important in the sensitization to TRAIL. To specifically investigate the importance of inhibition of class II HDACs, cells were exposed to MC1568 and MC1575, two compounds that inhibit class II HDAC in maize ( 30, 31). Both compounds caused hyperacetylation of tubulin but not of histone H3 and H4 in Jurkat ( Fig. 2A, lanes 6 and 7) and K562 cells ( Fig. 2B, lanes 7-9), supporting the notion that they also act as specific inhibitors of class II HDACs in human cells. MC1568 or MC1575 did not potentiate apoptosis, assessed by processing of caspase-3 or PARP cleavage in Jurkat or K562 cells ( Fig. 2C and D, lanes 11-14). CHAP31, a broad range HDACi that does not inhibit HDAC6 ( 29, 36), caused hyperacetylation of histone H3 and H4 but not of tubulin ( Fig. 2A, lane 5 and B, lane 6). CHAP31 also potentiated TRAIL-induced apoptosis, assessed by phosphatidylserine externalization, caspase-3 processing, and PARP cleavage ( Fig. 2C and D, lanes 9 and 10). Taken together, these results showed that inhibition of class II HDACs, such as HDAC6, alone does not result in the potentiation of TRAIL-induced apoptosis.

Figure 2.
Figure 2.

Inhibition of class I but not class II HDAC is required for potentiation of TRAIL-induced apoptosis. A, Jurkat cells were incubated for 8 hours either alone (Con) or with TSA (0.25 μmol/L), depsipeptide (Dep, 10 nmol/L), MS-275 (MS, 2.5 μmol/L), CHAP31 (C31, 100 nmol/L), MC1568 (1568, 5 μmol/L), or MC1575 (1575, 10 μmol/L). B, K562 cells were incubated for 12 hours with either the same as in (A) or indicated concentration of the HDACi except CHAP31 (25 nmol/L). HDACi concentrations were used, which alone did not induce any apoptosis. Cell lysates were prepared and analyzed by Western blotting as described in Materials and Methods for either tubulin, acetylated tubulin, or acetylated histone H3 or H4 (Ac-H3 or Ac-H4). C, Jurkat cells were incubated for 8 hours and D, K562 cells were incubated for 12 hours with the above concentrations of HDACi (A and B). Following HDACi treatment, Jurkat and K562 cells were exposed to TRAIL (20 and 100 ng/mL, respectively) for a further 4 hours and then apoptosis was assessed by phosphatidylserine (PS) externalization, processing of caspase-3, and cleavage of PARP. No phosphatidylserine values are given for compounds MC1568 and MC1575 because their colors interfere with the method.

Inhibition of HDAC8 is not important for sensitization to TRAIL. First, we confirmed that depsipeptide acted as a specific class I HDACi ( 37) in Jurkat and K562 cells, as it caused accumulation of acetylated histones but not acetylated tubulin ( Fig. 2A and B, lane 3). Depsipeptide (10 nmol/L) potentiated TRAIL-induced apoptosis in Jurkat and K562 cells, assessed by phosphatidylserine externalization, caspase-3 processing, and PARP cleavage ( Fig. 2C and D, lanes 5 and 6). Furthermore, pretreatment for 12 hours with nontoxic concentrations of depsipeptide (1-10 nmol/L), CHAP31 (2.5-25 nmol/L), and TSA (0.025-2.5 μmol/L) caused a concentration- and time-dependent sensitization of K562 cells to TRAIL. 3 These data support the hypothesis that inhibition of class I HDACs rather than class II resulted in the sensitization to TRAIL-induced apoptosis. MS-275, a promising HDACi, inhibits tumor growth in vivo and induces apoptosis following an early increase in reactive oxygen species, perturbation of mitochondria, and activation of the intrinsic pathway ( 38). MS-275 preferentially inhibits HDAC1 (IC50 ∼0.3 μmol/L) compared with HDAC3 (IC50 ∼8 μmol/L) and does not inhibit HDAC8 (IC50 > 100 μmol/L; ref. 32). MS-275 (1-2.5 μmol/L) caused hyperacetylation of histones but not tubulin in Jurkat and K562 cells ( Fig. 2A, lane 4 and B, lanes 4 and 5) consistent with inhibition of cellular HDACs. Pretreatment of Jurkat or K562 cells with MS-275 (2.5 μmol/L) potentiated TRAIL-induced apoptosis ( Fig. 2C and D, lanes 7-8). Furthermore, pretreatment of K562 cells with MS-275 (0.1-25 μmol/L) alone did not induce apoptosis but caused a concentration-dependent sensitization to TRAIL ( Fig. 3A ). Inhibition of HDAC1 may be more important than inhibition of HDAC3, as low MS-275 concentrations (1 μmol/L) sensitized K562 cells to apoptosis ( Fig. 3A). To confirm that MS-275 was selectively inhibiting HDAC1 and HDAC2 rather than HDAC3, K562 cells were exposed to MS-275 or depsipeptide followed by immunoprecipitation of HDAC1-3 and assessment of their activity. Following immunoprecipitation of HDAC1 or HDAC2, Western blot analysis revealed the presence of both HDAC1 and HDAC2 but not HDAC3 (data not shown) compatible with HDAC1 and HDAC2 being present in the same stable multicomponent cellular complexes, such as the Sin3, NuRD, and coREST nuclear complexes ( 11, 39). Immunoprecipitation of HDAC3 revealed the presence of HDAC3 but not HDAC1 or HDAC2 compatible with HDAC3 existing in distinct multisubunit complexes from HDAC1 or HDAC2 and these complexes often associate directly with the corepressors NCoR and SMRT ( 11, 39). Depsipeptide (10 nmol/L) completely inhibited HDAC1 and HDAC2 activity ( Fig. 3B, lanes 2 and 6). MS-275 (1-2.5 μmol/L) inhibited activities of HDAC1 and HDAC2 by ∼50% to 70% ( Fig. 3B, lanes 3, 4, 7, and 8). However, MS-275 (1 μmol/L) did not inhibit HDAC3 ( Fig. 3B, lane 11) but sensitized to TRAIL, supporting the suggestion that inhibition of HDAC3 is not required for sensitization to TRAIL-induced apoptosis. However, we cannot exclude that inhibition of HDAC3 enhances sensitization to TRAIL because higher concentrations of MS-275 (2.5 μmol/L) inhibit HDAC3 activity (∼20%; Fig. 3B, lane 12) and caused a greater sensitization to TRAIL.

Figure 3.
Figure 3.

Inhibition of HDAC8 does not sensitize cells to TRAIL-induced apoptosis. A, K562 cells were incubated for 12 hours with the indicated concentrations of MS-275. Cells were then exposed to TRAIL (100 ng/mL) for a further 4 hours and apoptosis was assessed by phosphatidylserine externalization. Columns, mean of three determinations; bars, SD. B, K562 cells were exposed for 12 hours to depsipeptide (10 nmol/L) or the indicated concentration of MS-275 followed by immunoprecipitation (IP) with an antibody to HDAC1, HDAC2, or HDAC3. The activity of the immunoprecipitated HDAC was then measured as described in Materials and Methods. Columns, mean of three separate experiments expressed as percentage of the control HDAC activity; bars, SD. C, Jurkat cells were exposed for 8 hours to the indicated concentrations of SB-556629 or SB-379278A and then examined for either acetylated histone H3 or H4. D, Jurkat cells were exposed for a further 4 hours to TRAIL (20 ng/mL) and apoptosis was assessed by phosphatidylserine externalization or processing of caspase-3.

Taken together, these results suggest that inhibition of HDAC8, and possibly also inhibition of HDAC3, is not required for the sensitization to TRAIL. To further exclude a possible role for HDAC8, Jurkat cells were exposed to SB-379278A, a specific potent inhibitor of HDAC8 (IC50 ∼0.5 μmol/L; ref. 32). SB-379278A (1-50 μmol/L) did not cause hyperacetylation of histone H3 and H4 ( Fig. 3C, lanes 7-9), demonstrating that even these high concentrations did not inhibit HDAC1-3 in agreement with previous work ( 32). Importantly, SB-379278A (1-50 μmol/L) did not potentiate TRAIL-induced apoptosis in Jurkat cells, assessed by phosphatidylserine externalization or processing of caspase-3 ( Fig. 3D, lanes 11-16). Clearly, inhibition of HDAC8 is not required for potentiation of TRAIL-induced apoptosis. A novel HDACi, Scriptaid, also known as SB-556629, and structurally related to TSA, inhibits all three class I HDACs tested with an IC50 of ∼0.6 μmol/L for HDAC1 and HDAC3 and of ∼1 μmol/L for HDAC8 ( 32, 40). In Jurkat cells, SB-556629 caused a concentration-dependent accumulation of acetylated histone H3 and H4, demonstrating inhibition of cellular HDACs even at the lowest concentration used (0.1 μmol/L; Fig. 3C, lanes 3-6). Low concentrations of SB-556629 (0.5 μmol/L), which alone cased minimal apoptosis, markedly potentiated TRAIL-induced apoptosis, assessed by both phosphatidylserine externalization and processing of caspase-3 ( Fig. 3D, lanes 8 and 10). Taken together, these data show that inhibition of some but not all class I HDACs result in a sensitization to TRAIL. Our data show that inhibition of HDAC8, and possibly also of HDAC3, does not sensitize to TRAIL-mediated apoptosis. Thus, inhibition of HDAC1 and HDAC2 seem to be most important for the sensitization.

HDAC1 and HDAC2 but not HDAC3 is important for sensitization to TRAIL. To further define which HDAC is important for HDACi-mediated sensitization to TRAIL, we used siRNA to selectively knockdown HDAC1-3. In preliminary experiments in K562 cells, we failed to obtain a good knockdown of HDAC1-3 probably because of a low transfection efficiency of suspension cells. We therefore used human prostatic DU145 tumor cells, partly because the expression of HDAC1-8 in these cells is similar to the profile in CLL cells (data not shown) and as adherent cells they are more efficiently transfected. Depsipeptide (10 nmol/L) sensitized DU145 cells to TRAIL-induced apoptosis through both TRAIL-R1 and TRAIL-R2 signaling pathways, as the cells were sensitized to both HGS-ETR1 and HGS-ETR2 ( Fig. 4A ), agonistic antibodies for both TRAIL-R1 and TRAIL-R2, respectively ( 26, 41). All three oligonucleotides for HDAC3 (HDAC3a-c) and two oligonucleotides for HDAC1 (HDAC1b and 1c) and HDAC2 (HDAC2a and 2c) resulted in a marked reduction in protein levels of the corresponding HDAC ( Fig. 4B). The effect of all oligonucleotides seemed specific, as knockdown of any particular HDAC did not result in a decrease in other HDACs; for example, knockdown of HDAC3 did not result in any knockdown of HDAC1, HDAC2, or HDAC6 ( Fig. 4B, lanes 8-10). To overcome the reported toxicity of knockdown of class I HDACs ( 42), cells were exposed to siRNA for short periods up to 36 hours to minimize toxicity, and then treated with TRAIL (100 ng/mL) for 4 hours. Knockdown of both HDAC1 and HDAC2 sensitized to TRAIL whereas efficient knockdown of HDAC3 had no effect on TRAIL-induced apoptosis ( Fig. 4C). Sensitization only occurred with the siRNAs that caused a decrease in expression of HDAC1 and HDAC2 and not with the siRNAs that did not cause a loss in protein expression ( Fig. 4B and C). These data clearly show that inhibition of HDAC3 is not required for the sensitization to TRAIL-induced apoptosis and also support the suggestion that inhibition of HDAC1 and/or HDAC2 is important for sensitization. Knockdown of HDAC1 and HDAC2 clearly sensitized DU145 cells to TRAIL-induced apoptosis as the increase in phosphatidylserine externalization was accompanied by the processing of capase-3 to its catalytically active large subunits (p20, p19, and p17; Fig. 4D, lanes 6 and 10), whereas those oligonucleotides (1a and 2b) that did not result in a decrease in their corresponding HDACs did not cause an increase in caspase-3 processing ( Fig. 4D, lanes 4 and 8). Cleavage of PARP was only observed in those cells with complete processing of caspase-3 to its p19/p17 subunits, demonstrating that the processed caspase-3 was catalytically active ( Fig. 4D, lanes 6 and 10).

Figure 4.
Figure 4.

Knockdown of HDAC1 and HDAC2 but not HDAC3 sensitizes DU145 cells to TRAIL-induced apoptosis. A, DU145 cells were pretreated with depsipeptide (10 nmol/L) for 6 hours then exposed for a further 4 hours to TRAIL (100 ng/mL), HGS-ETR1 (500 ng/mL), or HGS-ETR2 (500 ng/mL). Apoptosis was assessed by phosphatidylserine externalization. DU145 cells were exposed for 36 hours to the indicated siRNA oligonucleotide (20 nmol/L) and B, cell lysates were prepared and analyzed by Western blotting using either HDAC1, HDAC2, HDAC3, or HDAC6 antibodies. Following exposure to the oligonucleotides, cells were also exposed for a further 4 hours to TRAIL (100 ng/mL) and either C, apoptosis was assessed by phosphatidylserine externalization or D, cell lysates were prepared and analyzed by Western blotting for the processing of caspase-3 and the cleavage of PARP. Columns (A and C), mean of three experiments; bars, SD.

Although our data with HDACi ( Fig. 2C and D) suggested that inhibition of HDAC6 is not required for the sensitization to TRAIL, we wished to confirm this with direct genetic evidence because of the interest in HDAC6, as both a microtubule-associated deacetylase and an HSP90 deacetylase, and its role in aggresome formation ( 35, 43, 44). All three oligonucleotides for HDAC6 caused a marked decrease in HDAC6 protein levels without affecting levels of HDAC1-3 ( Fig. 5A ). The decrease of HDAC6 protein was accompanied by accumulation of acetylated tubulin, demonstrating that the knockdown was functionally effective ( Fig. 5A, lanes 3-5) and supporting previous suggestions that HDAC6 is the major cellular tubulin deacetylase ( 34, 35). However, despite the knockdown of HDAC6, no sensitization to TRAIL-induced apoptosis was observed ( Fig. 5B). An oligonucleotide for HDAC1, used as a positive control, again caused a knockdown of HDAC1 and sensitized to TRAIL ( Fig. 5). Taken together with the inhibitor data, our results show that inhibition of HDAC6 is not required for HDACi-mediated sensitization to TRAIL.

Figure 5.
Figure 5.

Knockdown of HDAC6 does not sensitize DU145 cells to TRAIL. DU145 cells were exposed for 36 hours to the indicated siRNA oligonucleotide (20 or 30 nmol/L for HDAC1 or HDAC6, respectively). A, cell lysates were prepared and analyzed by immunoblotting with anti-HDAC1, HDAC2, HDAC3, HDAC6, or acetyl α-tubulin antibody. Following exposure to the oligonucleotides, cells were also exposed for a further 4 hours to TRAIL (100 ng/mL). B, apoptosis was assessed by phosphatidylserine externalization. Columns, mean of three separate experiments; bars, SD.

Inhibition of HDAC class I but not class II sensitizes CLL cells to TRAIL. To choose which HDACi may be most useful clinically in combination with TRAIL for the treatment of CLL, we wished to confirm that the HDACi exerting the greatest effects in cell lines showed similar effects in primary CLL cells. CLL cells were exposed to various HDACi that showed the greatest range of activities and examined for inhibition of class I and II HDACs. Based on the results obtained with acetylated tubulin and acetylated histone H3 and H4, TSA inhibited class I and II HDACs, depsipeptide and MS-275 inhibited class I and MC1568 inhibited class II HDACs ( Fig. 6A ). These results were virtually identical to those obtained with Jurkat and K562 cells ( Fig. 2). Pretreatment with depsipeptide and MS-275 but not SB-379278A sensitized CLL cells to TRAIL, as assessed by phosphatidylserine externalization ( Fig. 6B). Further support that this sensitization in CLL cells by depsipeptide and MS-275 but not SB-379278A and MC1568 was due to apoptosis was provided by the increased processing of both the initiator caspase-8 to its partially processed p43/p41 fragments and its p18 catalytically active large subunit and also caspase-3 to its catalytically active large subunits, p19/p17, as well as increased cleavage of PARP ( Fig. 6C compare lanes 4 and 6 to the other lanes). A range of MS-275 concentrations was used, as this HDACi induces apoptosis in CLL cells over prolonged time periods ( 45). In the present study, increasing concentrations of MS-275 (0.25-2.5 μmol/L) also induced a small amount of apoptosis above spontaneous levels but a clear sensitization to TRAIL was also observed ( Fig. 6B and C). Thus, in CLL cells, inhibition of HDAC class I but not class II is required for sensitization; within class I, inhibition of HDAC8 is not required. These results are in excellent agreement with the results in cell lines.

Figure 6.
Figure 6.

Inhibition of HDAC class I but not class II sensitizes CLL cells to TRAIL. A, CLL cells were incubated for 16 hours either alone (Con) or with TSA (0.25 μmol/L), depsipeptide (10 nmol/L), MS-275 (0.25-2.5 μmol/L), SB-379278A (SB; 50 μmol/L), or MC1568 (5 μmol/L), and lysates were analyzed by Western blotting for either tubulin, acetylated tubulin, or acetylated histone H3 or H4. CLL cells were pretreated for 16 hours with the HDACi as indicated in (A) and then exposed to TRAIL (100 ng/mL) for a further 4 hours and apoptosis was assessed by phosphatidylserine externalization (B). Columns, mean from eight individual patients; bars, SE. *, P < 0.05, significantly greater than its corresponding control (paired one-tailed t test). C, after exposure to TRAIL, cells were also analyzed for the processing of caspase-8, caspase-3, and cleavage of PARP by Western blotting. In the experiment on caspase processing, the same concentrations of HDACi indicated in (A) were used except that only MS-275 (2.5 μmol/L) was used. D, CLL cells were pretreated for 16 hours with the indicated concentration of sodium valproate and then exposed to TRAIL (100 ng/mL) for a further 4 hours and apoptosis was assessed by phosphatidylserine externalization. Columns, mean from nine individual patients; bars, SE. *, P < 0.01, significantly different from the control (paired one-tailed t test). Valproate alone (0.25-2.5 mmol/L) did not induce any apoptosis above control values (data not shown). Depsipeptide (10 nmol/L), used as a positive control, sensitized cells to TRAIL similar to the data in (B).

We have previously shown that CLL cells could be sensitized to TRAIL by sodium valproate, a widely used anticonvulsant drug that also has activity as an HDACi ( 26, 46). In our previous study to obtain proof of principle, we used high concentrations of valproate (2 mmol/L; ref. 26) above the normal clinical range (0.3-0.6 mmol/L) used in the management of epilepsy ( 47). Hence, we examined a range of valproate concentrations. Pretreatment of CLL cells for 16 hours with valproate (0.25-2 mmol/L) caused a concentration-dependent sensitization to TRAIL-induced apoptosis ( Fig. 6D).

Discussion

To gain insight as to which individual HDACs have to be inhibited to sensitize to TRAIL, we used HDACi with different specificities. Such information is important mechanistically as well as having significant clinical implications. We show unequivocally that inhibition of class II HDACs is not required for the sensitization to TRAIL, based on the lack of effects of knockdown of HDAC6 together with our findings that class II inhibitors, MC1575 and MC1568 ( 30, 31), which inhibit both HDAC4 and HDAC6, 4 do not sensitize Jurkat, K562, and CLL cells to TRAIL ( Figs. 2, 5, and 6). Our data also suggest that acetylation of tubulin and Hsp90 is not required for the sensitization, as HDAC6 is the major HDAC regulating acetylation of these molecules ( 34, 35, 43, 44).

Of the four class I HDACs, our data showing that SB-379872A, a specific inhibitor of HDAC8 ( 32), does not potentiate TRAIL-induced apoptosis showed that HDAC8 is not required for the potentiation. Support for this conclusion was provided by the finding that low concentrations of MS-275, which preferentially inhibits HDAC1 compared with HDAC3 and does not inhibit HDAC8 ( 32), sensitized TRAIL-induced apoptosis in K562 and CLL cells ( Figs. 3A and 6B). These findings also implicated inhibition of HDAC1 rather than HDAC3 in the sensitization, but we could not exclude HDAC2, as it was not examined in the study of Hu et al. ( 32). Based on studies solely with inhibitors, we could not completely exclude the importance of inhibition of HDAC3, as there is no specific inhibitor available. However, a possible role for HDAC3 was largely excluded as extensive knockdown of HDAC3 did not sensitize DU145 cells to TRAIL ( Fig. 4C and D). However, in contrast, knockdown of HDAC1 and HDAC2 markedly sensitized DU145 cells to TRAIL ( Fig. 4C and D). Sensitization to TRAIL-induced apoptosis, as assessed by phosphatidylserine externalization, either by inhibition of HDAC class I with HDACi or knockdown of HDAC1 or HDAC2 with siRNA, was in all cases accompanied by the complete processing of caspase-3 to its p20, p19, and p17 large subunits ( Figs. 2, 4D, and 6C), which was active as it was accompanied by PARP cleavage. Caspase-3 is initially processed at Asp175, between its large and small subunits, to yield its p20/p12 subunits ( 48). The p20 subunit then normally undergoes autocatalytic processing to yield its p19/17 products but this autoprocessing may be inhibited in some cells, such as those expressing high levels of XIAP ( 48). This inhibition may be relieved by the mitochondrial release of proapoptotic molecules, such as Smac ( 48, 49). In this regard, it was of interest to note that knockdown of HDAC3 ( Fig. 4D, lanes 12 and 14) and inhibition of HDAC class II in K562 cells ( Fig. 2D, lanes 12 and 14) followed by TRAIL treatment resulted in partial processing of caspase-3 to its p20 form without PARP cleavage or phosphatidylserine externalization. Our data suggest that the p20 form of caspase-3 in these cells was catalytically inactive, most probably because it was bound to XIAP. The ability of some HDACi to perturb mitochondria is well known as HDACi alone induce apoptosis by activation of the intrinsic pathway ( 14, 15, 17). However, HDACi-mediated sensitization of cells to TRAIL-induced apoptosis occurs via the extrinsic pathway with caspase-8 as the apical caspase and does not necessarily require mitochondrial involvement ( 24). Thus, the p20 form of caspase-3 observed in our experiments may be a consequence of a small amount of caspase-8 activation resulting in an initial cleavage of caspase-3 but without any mitochondrial perturbation to release Smac and relieve XIAP-mediated inhibition. Further studies may help resolve the mechanism whereby knockdown of HDAC3 results in a small activation of caspase-3 processing as well as the contribution of mitochondrial perturbation to HDACi-mediated sensitization to TRAIL.

Although the results with the knockdown of HDAC1 and HDAC2 seem unequivocal, some reservations have to be expressed. Effects of knockdown of HDACs are not equivalent to inhibition of enzyme activity. HDACi but not knockdown of HDAC1, HDAC2, or HDAC3 results in the accumulation of total acetylated histone H3 and H4, although knockdown of HDAC1 and HDAC3 results in the accumulation of acetylated H3K18 and acetylated H3K9, respectively ( 50). Also, knockdown of one HDAC may affect the activity of another HDAC, particularly if they are in the same multiprotein complexes, such as the presence of HDAC1 and HDAC2 in Sin3, NuRD, and CoREST complexes ( 39). Despite these reservations, our data strongly suggests that HDAC1 and HDAC2 are the major targets for the HDACi-mediated sensitization to TRAIL-induced apoptosis.

Using a larger cohort of patients (n = 28) than in the present study, we have previously observed that depsipeptide-mediated sensitization to TRAIL-induced apoptosis occurred independently of both p53 and IGHV status and resistance to fludarabine ( 26). Similarly, the present cohort of nine patients also included individuals sensitive or resistant to fludarabine and of both mutated/unmutated IGHV status. Cells from all these individuals were sensitized to TRAIL-induced apoptosis by either depsipeptide or valproate ( Fig. 6C and D). Thus, the present data in agreement with our previous reports ( 24, 26, 27) support the possibility of using a combination of an HDACi and TRAIL in the treatment of CLL and possibly other lymphoid malignancies. Recently, we highlighted that this sensitization occurred by signaling through TRAIL-R1 and not TRAIL-R2 ( 26, 27), thereby necessitating the use of a form of TRAIL that signals through TRAIL-R1, such as a TRAIL-R1 agonistic antibody or a mutant form of TRAIL that signals specifically through TRAIL-R1 ( 27). In the present study, we have tried to ascertain the critical properties required of an HDACi to sensitize to TRAIL-induced apoptosis. We have shown that class I and not class II HDACs are involved in the sensitization and within class I HDACs, inhibition of HDAC1 and HDAC2 but not HDAC3 and HDAC8 is critical for sensitization. In this regard, none of the HDACi that have recently entered phase I and II clinical trials was developed to target specific HDAC isoforms ( 9, 12). Compounds that inhibit other HDACs, such as HDAC6, could lead to unnecessary host toxicity without any increased sensitization, as sensitization only requires inhibition of HDAC1 and/or HDAC2. However, we cannot exclude that other properties of these HDACi may have beneficial antitumor effects. Based on the present study, the optimal HDACi for sensitization to TRAIL in CLL would be a relatively specific compound that inhibits HDAC1 and/or HDAC2 but not other HDACs, such as depsipeptide, MS-275, and SB-429201 ( 32, 37). We have also shown that clinically achievable concentrations of the anticonvulsant sodium valproate can sensitize CLL cells to TRAIL. Thus, for a clinical trial in CLL, we propose the combination of a form of TRAIL that signals through TRAIL-R1 together with either sodium valproate or preferably a more selective inhibitor of HDAC1/2.

Acknowledgments

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. E. Sausville, M. Yoshida, and E. Hu for reagents, and Drs. M. Mulheran, K. Cain, and L. Altucci for help with the statistical analysis and the HDAC activity assays.

Footnotes

  • ↵3 Unpublished data.

  • ↵4 L. Altucci, personal communication.

  • Received December 21, 2005.
  • Revision received April 21, 2006.
  • Accepted April 27, 2006.

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Cancer Research: 66 (13)
July 2006
Volume 66, Issue 13

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Inhibition of Histone Deacetylase Class I but not Class II Is Critical for the Sensitization of Leukemic Cells to Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis
Satoshi Inoue, Antonello Mai, Martin J.S. Dyer and Gerald M. Cohen
Cancer Res July 1 2006 (66) (13) 6785-6792; DOI: 10.1158/0008-5472.CAN-05-4563
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