Interactions between the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) and the cyclin-dependent kinase (CDK) inhibitor flavopiridol (FP) were examined in human leukemia cells. Simultaneous exposure (24 h) of myelomonocytic leukemia cells (U937) to SAHA (1 μM) and FP (100 nM), which were minimally toxic alone (1.5 ± 0.5% and 16.3 ± 0.5% apoptosis respectively), produced a dramatic increase in cell death (ie 63.2 ± 1.9% apoptotic), reflected by morphology, procaspase-3 and -8 cleavage, Bid activation, diminished ΔΨm, and enhanced cytochrome c release. FP blocked SAHA-mediated up-regulation of p21CIP1 and CD11b expression, while inducing caspase-dependent Bcl-2 and pRb cleavage. Similar interactions were observed in HL-60 and Jurkat leukemic cells. Enhanced apoptosis in SAHA/FP-treated cells was accompanied by a marked reduction in clonogenic surivival. Ectopic expression of either dominant-negative caspase-8 (C8-DN) or CrmA partially attenuated SAHA/FP-mediated apoptosis (eg 45 ± 1.5% and 38.2 ± 2.0% apoptotic vs 78 ± 1.5% in controls) and Bid cleavage. SAHA/FP induced-apoptosis was unaffected by the free radical scavenger L-N-acetyl cysteine or the PKC inhibitor GFX. Finally, ectopic Bcl-2 expression marginally attenuated SAHA/FP-related apoptosis/cytochrome c release, and failed to restore clonogenicity in cells exposed to these agents. Together, these findings indicate that SAHA and FP interact synergistically to induce mitochondrial damage and apoptosis in human leukemia cells, and suggest that this process may also involve engagement of the caspase-8-dependent apoptotic cascade.
Suberoylanilide hydroxamic acid (SAHA) a is second-generation polar–planar compound that is approximately 1000-fold more potent on a molar basis than hexamethylenebisacetamide (HMBA) in inducing maturation in murine erythroleukemia cells (MEL).1 SAHA, like sodium butyrate,2 trichostatin A,3 depsipeptide,4 and MS-2755 acts as an inhibitor of histone deacetylase.1,6 Acetylation of histones results in conformational changes that permit transcriptional activation of genes involved in diverse cellular processes, including differentiation.7 In MEL cells, SAHA-induced differentiation is associated with cell cycle arrest and induction of the cyclin-dependent kinase inhibitor p21CIP1.8 However, while SAHA also induces p21CIP1 expression in human leukemia cells such as U937 and HL-60, its ability to induce maturation in these cell lines may be limited by its capacity to trigger mitochondrial damage and apoptosis.9 In view of these and other characteristics, histone deacetylase inhibitors such as SAHA have become the focus of interest as antineoplastic agents,10 and phase I trials of SAHA have recently been initiated in humans.
Flavopiridol (FP; L86 8175; NSC 649890) is a synthetic flavone derived from the flavanoid rohitukine which is a potent inhibitor of cyclin-dependent kinases (CDKs) 1, 2, 4, 6 and 5.11,12 It binds tightly to the ATP-binding domain of CDKs, thereby blocking activation of these enzymes.13 In preclinical studies, FP has been shown to be a potent inducer of apoptosis in cells of both hematopoietic14 and non-hematopoietic15 origin. FP is the first of the CDK inhibitors to undergo phase I/II evaluation in humans,16 and plasma concentrations in excess of those displaying cytotoxicity in vitro (eg 300 nM) are achievable.17 Preclinical evidence also indicates that FP-related cell cycle perturbations can potentiate apoptosis induced by conventional cytotoxic agents.18
In a recent communication,19 we reported that co-administration of FP with the tumor-promoting PKC activator phorbol 12-myristate 13-acetate (PMA) did not enhance leukemic cell maturation; instead, FP induced multiple perturbations in cell cycle-related events, including down-regulation of the CDKI p21CIP1, that culminated in a marked potentiation of mitochondrial dysfunction and apoptosis. In view of evidence that SAHA is also a potent inducer of p21CIP1 in leukemia cells,8,9 and that in human leukemia cells it primarily triggers apoptosis rather than maturation,9 the question of how SAHA might interact with FP arose. To address this issue, we have examined the effects of simultaneous administration of the SAHA with FP in human leukemia cells. Here, we report that administration of a pharmacologically relevant concentration of FP (eg 100–150 nM) with a subtoxic concentration of SAHA (eg ⩽2 μM) leads to multiple perturbations in cell cycle regulation, including abrogation of p21CIP1 induction and pRb degradation, impaired maturation, and marked mitochondrial dysfunction (eg cytochrome c release; loss of mitochondrial membrane potential), caspase activation, and apoptosis. Moreover, synergistic induction of apoptosis by FP and SAHA occurs in several human myeloid and lymphoid leukemic cell types, may proceed in part via engagement of the extrinsic apoptotic cascade, and is associated with a marked reduction in clonogenic potential. Together, these findings suggest that the CDK inhibitor FP interacts with the HDI SAHA in a manner that promotes cell cycle dysregulation, mitochondrial damage, and caspase activation, culminating in the synergistic induction of apoptosis. They also raise the possibility that combining CDK inhibitors with HDIs such as SAHA may warrant further examination as a novel antileukemic strategy.
Materials and methods
The myelomonocytic leukemia cell line U937 was derived from a patient with histiocytic lymphoma and was obtained from ATCC (Rockville, MD, USA). HL-60 promyelocytic leukemia cells, and Jurkat T-lymphoblastic leukemia cells were also purchased from ATCC. Cells were cultured and maintained in logarithmic growth phase in RPMI 1640 medium supplemented with sodium pyruvate, MEM essential vitamins, L-glutamate, penicillin, streptomycin, and 10% FBS (Gibco, Life Technologies, Grand Island, NY, USA). Cells were maintained in a 37°C, 5% CO2, fully humidified incubator and passed twice weekly.
Transfectant cell lines
U937 cells were transfected by electroporation (264 V, 750 mF, R9). Transfectant U937 leukemia cells stably overexpressing the antiapoptotic protein Bcl-2 were obtained as reported previously.20 These cells, designated U937/Bcl-2 cells, were generated along with their empty vector counterparts (ie U937/pCEP4), maintained as described above in the presence of hygromycin B (Roche Diagnostic, Indianapolis, IN, USA) (400 μg/ml), and transferred to selection-free media 24 h before experimentation. CrmA-expressing cell lines and dominant-negative caspase-8 were obtained by transfecting U937 cells with CrmA/pcDNA 3.1 and dominant-negative caspase 8/pcDNA 3.1 (kindly provided by Dr Kapil Bhalla, H Lee Moffitt Cancer Center, Tampa, FL, USA) cloned into pcDNA3.1 (Invitrogen Corporation, Carlsbad, CA, USA). U937 cells stably transfected with a p21CIP1 antisense construct were obtained as previously described.21 Empty vector transfectants served as negative controls. Clonal populations were established by limiting dilution. Transfectant cell lines were maintained in the presence of G418 (Gibco) (400 μg/ml). All experiments were performed utilizing cells in logarithmic phase growth.
Drugs and chemicals
SAHA was kindly provided by Dr Victoria Richon (Memorial Sloan-Kattering Cancer Center, New York, NY, USA). Stock solutions were prepared in DMSO (Sigma Chemical, St Louis, MO, USA) at a concentration of 10−2 M and dissolved in PBS prior to use. Flavopiridol (L86 8275; NCS 649890) was kindly provided by Dr Edward Sausville (Cancer Treatment and Evaluation Program, National Cancer Institute, Bethesda, MD, USA). FP was formulated in DMSO as 10−2 M stock solutions and stored at −20°C. Cells in logarithmic-phase growth were suspended at 2.5 × 105 cells/ml and exposed to test agents for various intervals as indicated. In experiments involving examination of reactive oxygen species (ROS), cells were pretreated with L-N-acetylcysteine (L-NAC) (Sigma) 0.5 to 1 h before the addition of lactacystin (BioMol, Plymouth Meeting, PA, USA),21 SAHA, FP or SAHA and FP in combination. Bryostatin 1 was provided by Dr AJ Murgo, CTEP-NCI, and was added 0.5 h after lactacystin treatment.
The PKC inhibitor bisindoylmaleimide I (GF109203X) (Calbiochem, San Diego, CA, USA) was formulated in DMSO, added 0.5 h before the addition of SAHA and FP. The pan-caspase inhibitor BOC-Asp(Ome)-FMK was purchased from Enzyme System Products (Livermore, CA, USA) and dissolved in DMSO. Human recombinant TNF (Calbiochem) and TNF soluble receptor (R&D Systems, Minneapolis, MN, USA) were dissolved in PBS + 0.5% BSA. Cycloheximide was purchased from (Sigma), dissolved in PBS, and stored at −20°C.
Morphological assessment of apoptosis
Apoptotic cells were evaluated by morphological assessment of Wright–Giemsa-stained cytospin preparations. Apoptotic cells were identified by classical morphological features (ie nuclear condensation, cell shrinkage and formation of apoptotic bodies). Five or more randomly selected fields, encompassing a total of 1000 cells/slide, were evaluated to determine the percentage of apoptotic cells for each treatment condition as previously described.22
Annexin V/PI assay for apoptosis
Annexin V/PI (BD PharMingen, San Diego, CA, USA) analysis of cell death was carried out as per the manufacturer's instructions. In studies involving TNF and TNF soluble receptor, both compounds were combined in sterile medium and maintained at room temperature for 20 min prior to use. In these studies, 1–2 × 105 cells were harvested for each experimental condition. Analysis was carried out using a Becton Dickinson FACScan cytofluorometer (Mansfield, MA, USA).
Treated cells were lysed and equivalent quantities assayed per the manufacturer's instructions (Caspase-3/CP322 Assay Kit; BioVision, Palo Alto, CA, USA). The fold-increase in activity was calculated as the ratio between values obtained for treated samples vs those obtained in untreated controls.
Cell cycle analysis
Following drug treatment, cells were pelleted by centrifugation at 500 g × 6 min, and resuspended in 70% ethanol. The cells were incubated on ice for at least 1 h and resuspended in 1 ml cell cycle buffer (0.38 mM Na-Citrate, 0.5 mg/ml RNase A, and 0.01 mg/ml propidium iodide) at a concentration of 10 × 105 cells/ml, stored in the dark at 4°C until analysis (24 h), using a Becton Dickinson FACScan flow cytometer and Verity Winlist software (Verity Software, Topsham, ME, USA).
Expression of the monocytic differentiation marker CD11b was monitored by direct immunofluorescence staining and flow cytometric analysis as described previously.23 After drug treatment, suspension and adherent cells were enumerated using a Coulter Counter, and 2 × 106 cells were pelleted by centrifugation. The supernatant was aspirated, and the cells were resuspended in 300 μl of ice-cold PBS. Two 100-μl aliquots from each sample were then combined with either phycoerythrin-1 (10 μl) or the IgG control. Samples were incubated for 20 min at 4°C and diluted in PBS (1 ml). Sample data were collected using a Becton Dickinson FACScan flow cytometer and analyzed with Verity Winlist Software.
Assessment of mitochondrial membrane potential (ΔΨm)
At the indicated intervals, cells were harvested and 2 × 105 cells were incubated with 40 nM DiOC6 for 15 min at 37°C. Analysis was then carried out on a Becton Dickinson FACScan cytofluorometer. The percentage of cells exhibiting low levels of DiOC6, reflecting loss of mitochondrial membrane potential, was determined as previously described.21
Whole cell pellets were washed twice in PBS, resuspended in PBS, and lysed by the addition of 1 volume of loading buffer. Lysates were boiled for 10 min, centrifuged at 12 800 g for 5 min and quantified using Coomassie protein assay reagent (Pierce, Rockford, IL, USA). 25 μg of total proteins per point were separated by SDS-PAGE and electro-blotted to nitrocellulose. The blots were blocked in 5% non-fat milk in PBS-T and probed for 1 h with the appropriate dilution of primary antibody. Blots were washed 3 × 10 min in PBS-T and then incubated with a 1:2000 dilution of peroxidase-conjugated secondary antibody for 1 h at room temperature. Blots were again washed 3 × 10 min in PBS-T and then developed by enhanced chemiluminesence (New England Nuclear, Boston, MA, USA). Where indicated, blots were stripped and re-probed with antibodies directed against actin.
Antibodies for Western analysis
Primary antibodies for the following proteins were used at the designated dilutions: p21WAF1/CIP1 (Transduction Laboratories, Lexington, KY, USA) were used in a 1:1000 dilution; PARP (1:1000; BioMol); pro-caspase 3 (1:1000; Transduction Laboratories); cytochrome c (1:2000; BD PharMingen,); bcl-2 (1:2000; Dako, Glostrup, Denmark); caspase 8 (1:2000 Alexia Corporations, San Diego, CA, USA); bid (1:1000; Cell Signaling, Beverly, MA, USA); actin (1:2000; Sigma Chemicals). Secondary antibodies conjugated to horseradish peroxidase were obtained from Kirkegaard and Perry Laboratories (Gaithersburg, MD, USA).
Analysis of cytosolic cytochrome c
A previously described technique was employed.24 After treatment, cells (5 × 107/condition) were harvested by centrifugation at 600 g for 10 min at 4°C. The S-100, or cytosolic fraction, was prepared as described with minor modifications. Cell pellets were washed once with ice-cold phosphate-buffered saline and resuspended in 5 volumes of buffer A (20 mM Hepes–KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethlysufonyl fluoride, and 250 mM sucrose; all Sigma). After chilling for 30 min on ice, the cells were disrupted by 15 strokes of a Potter–Elvehjem (PTFE-pestle) homogenizer. The homogenate was then centrifuged twice to remove unbroken cells and nuclei (3000 g for 10 min at 4°C). The resulting supernatant was further centrifuged at 100 000 g for 1 h at 4°C, and the supernatants, designated as S-100 fractions, were immediately subjected to Western analysis as described above. For each condition, 25 μg of the S-100 fraction was loaded on the gel, and probed with the corresponding antibody.
Clonogenic survival assays
Following drug treatment, cell densities were determined using a Coulter Counter (Coulter Electronics, Hialeah, FL, USA). Cells were pelleted by centrifugation at 400 g for 6 min at room temperature, washed three times, and suspended in fresh medium to achieve a final cell density of 3 × 105 cells/ml. A total of 5000 cells/condition were plated in soft agar as described previously.24 Colonies, defined as groups of ⩾50 cells, were scored after 8 days of incubation. Clonogenic survival was expressed as the ratio of colonies formed by treated vs control cells x the ratio of cell densities at the end of drug exposure interval.
The significance of the differences between experimental conditions was determined utilzing the Student's t-test for unpaired observations. Analysis of synergism and antagonism was carried out using Median Dose Effect analysis in conjunction with a commercially available software program (Calcusyn; Biosoft, Ferguson, MO, USA).
SAHA and FP interact synergistically to induce apoptosis in multiple human leukemic cell lines
To assess the effect of FP administration on the response of leukemic cells to SAHA, U937 cells were exposed for 24 h to 0.5–2 μM SAHA in the presence or absence of 75–150 nM FP, after which the extent of apoptosis was determined by morphologic assessment of Wright–Giemsa-stained cytospin preparations (Figure 1a).While exposure to SAHA alone at concentrations up to 2.0 μM exerted minimal effects, consistent with our earlier findings,9 and exposure to FP alone was marginally toxic (ie inducing apoptosis in 7–18% of cells), combined exposure resulted in apoptosis in virtually 100% of cells, particularly at SAHA concentrations ⩾1 μM. Essentially equivalent results were obtained when apoptosis was monitored by the Annexin/PI assay (data not shown). A time course study revealed that potentiation of apoptosis in cells exposed to SAHA and FP was first noted at 6 h, and increased progressively over the ensuing 18 h (Figure 1b). Median Dose Effect analysis of apoptosis induction in cells exposed to FP and SAHA revealed combination index (CI) values considerably less than 1.0, corresponding to a highly synergistic interaction (Figure 1c). Finally, clonogenic assays revealed that whereas a 24-h exposure of cells to SAHA was only modestly inhibitory to the growth of clonogenic cells, and flavopiridol somewhat more so, combined treatment with both agents resulted in nearly a two-log reduction in colony formation (Figure 1d).
As shown in Figure 2a, co-administration of SAHA (1.0 and 1.5 μM) and FP (100 or 150 nM) for 24 h resulted in a marked potentiation of apoptosis as well as mitochondrial damage (ie loss of ΔΨm) in human promyelocytic leukemia cells, most notably at a FP concentration of 150 nM (HL-60; Figure 2b). These events were accompanied by enhanced cleavage/activation of procaspase-3 (Figure 2c). In addition, potentiation of SAHA-mediated lethality by FP was observed in T-lymphoblastic Jurkat cells (Figure 2d). Together, these findings indicate that the synergistic induction of apoptosis by SAHA and FP is not restricted to U937 leukemic cells, although the extent of cell death may vary between cell lines.
To characterize further the lethal effects of SAHA and FP in U937 cells, flow cytometric analysis of annexin V/PI-stained cells was carried out (Figure 3). This method can distinguish between early apoptosis, in which cells stain positively for annexin V (FL1) vs late apoptosis, in which breakdown of the cell membrane results in PI positivity (FL3). The time course study shown in Figure 3a revealed a sharp increase in apoptosis in SAHA/FP-treated cells at 5 h, with cell death approaching 60% at 18–24 h. A 24 h exposure to 1.0 μM SAHA resulted in essentially no increase in the number of dead cells, whereas 100 nM FP resulted in a modest increase in early and late cell death (Figure 3b). However, combined treatment with SAHA + FP resulted in a major increase in cells positive for annexin V alone (∼30%) and both annexin V and PI (∼32%). These findings raise the possibility that the combination of FP and SAHA may kill leukemic cells at both early and late intervals. Finally, the pan-caspase inhibitor Boc-fmk, when co-adminstered at concentrations ⩾20 μM, substantially blocked annexin/PI-staining of SAHA/FP-treated cells (Figure 3c), consistent with the notion that caspases play a critical role in inducing the apoptotic phenotype.
SAHA and FP induce cytochrome c release in U937 cells through a caspase-independent process
Mitochondrial events involved in FP/SAHA-mediated lethality were next examined (Figure 4). Exposure to 100 nM FP alone resulted in a modest amount of cytochrome c release into the S-100 fraction at 12 and 24 h (Figure 4a), consistent with our previous findings.19 In contrast, SAHA alone (1.0 μM) was ineffective in triggering cytochrome c release. However, combined exposure to both agents resulted in a marked increase in redistribution of cytochrome c. In addition, co-administration of the broad caspase inhibitor BOC-D-fmk (20 μM) minimally attenuated cytochrome c release at 24 h, indicating that the latter event lies upstream of caspase activation. In contrast, co-administration of BOC-D-fmk significantly diminished the loss of ΔΨm in SAHA/FP-treated cells (P < 0.05 vs SAHA + FP alone), indicating that the latter event, unlike the redistribution of cytochrome c, represents a secondary, caspase-dependent phenomenon.
Combined treatment with SAHA and FP activates caspase-8 and Bid through a caspase-dependent process
Consistent with these results, 24 h exposure to 1.0 μM SAHA failed to increase activity of caspase-3, while a modest increase was observed in cells treated with 100 nM FP (Figure 5a). However, combined treatment resulted in a marked increase in caspase-3 activation, reflected by the appearance of a 17 kDa caspase 3 cleavage fragment, as well as in degradation of full-length PARP, a major caspase-3 substrate (Figure 5b). Moreover, while combined exposure to FP and SAHA for 24 h resulted in enhanced cleavage of procaspase-3, procaspase-8, and Bid (Figure 5c), these events were largely inhibited by BOC-D-fmk. While such an observation suggests that each of these events is caspase-dependent, the possibility that other proteases (eg cathepsin) might be involved cannot be excluded. These findings, in conjunction with those shown in Figure 4a, indicate that FP and SAHA induce cytochrome c release directly, rather than through activation of the extrinsic, caspase-8/Bid-mediated pathway.
Combined treatment with SAHA and FP disrupts the cell cycle profile and blocks maturation of U937 cells
Effects of SAHA and FP on the cell cycle traverse of U937 cells are illustrated by the DNA histograms shown in Figure 6a. A 24 h exposure to 1.0 μM SAHA had essentially no effect on the cell cycle distribution of U937 cells, whereas 100 nM FP resulted in modest declines in the G0G1 and S-phase populations, accompanied by a reciprocal increase in the sub-diploid (apoptotic) cell fraction. However, combined treatment with SAHA and FP resulted in a very striking disorganization of the cell cycle profile, as well as a marked increase in the sub-diploid population. As shown in Figure 6b, 36 or 72 h exposure to 1.0 μM SAHA resulted in a modest increase in expression of the monocytic maturation marker CD11b (ie in 25.6 ± 5.7% of cells), whereas 100 nM FP induced a smaller increase (ie in 11.7 ± 1.9% of cells). However, in cells exposed to both agents, expression of CD11b was equivalent to that of controls (ie 2.7 ± 0.9%). These findings indicate that co-administration of FP interferes with rather than potentiates SAHA-mediated maturation in U937 cells.
Co-administration of SAHA and FP results in the caspase-independent inhibition of p21CIP1 induction and the caspase-dependent cleavage of Bcl-2 and pRb
Previous studies indicated that FP blocked induction of p21CIP1 by the PKC activator PMA in human leukemia cells through a transcriptional rather than a caspase-mediated mechanism.19 To determine whether a similar phenomenon might occur in response to SAHA, U937 cells were exposed to 1.0 μM SAHA, 100 nM FP, or the combination in the presence or absence of BOC-D-fmk, after which p21CIP1 expression was monitored by Western analysis. As previously reported,19 SAHA alone induced p21CIP1 expression by 12 h, and to an even greater extent by 24 h (Figure 7a). Moreover, co-administration of FP, which by itself failed to induce p21CIP1, blocked up-regulation of this CDKI by SAHA, an effect that was not prevented by the caspase inhibitor BOC-D-fmk (Figure 7). This observation indicates that FP blocks SAHA-mediated up-regulation of p21CIP1 through a caspase-independent mechanism, analogous to findings involving PMA.19 Co-administration of SAHA and FP was also associated with enhanced cleavage of Bcl-2 into a putatively pro-apoptotic fragment,25 a phenomenon that was abrogated by BOC-D-fmk (Figure 7).
Co-administration of SAHA and FP resulted in a decrease in total pRb expression at 12 h, an effect that was substantially more pronounced by 24 h (Figure 7a). The decline in pRb expression in FP/SAHA-treated cells was abrogated by BOC-D-fmk, consistent with previous reports indicating that pRb is a substrate for apoptotic caspases.26 Both FP alone and the combination of FP + SAHA resulted in increased expression of underphosphorylated pRb, although this effect was presumably attenuated by pRb degradation. In support of this notion, addition of BOC-D-fmk to the SAHA/FP combination produced a marked increase in levels of underphosphorylated pRb.
To assess the functional significance of disruption of p21CIP1 induction by FP, a U937 cell line stably transfected with a p21CIP1 antisense construct was employed.21 As shown in Figure 7b, induction of p21CIP1 in control pREP cells by 1 μM SAHA was largely abrogated in the antisense line. This phenomenon was accompanied by a marked increase in SAHA-mediated apoptosis (Figure 7c), analogous to the phenomenon we have previously described in the case of sodium butyrate.27 Moreover, dysregulation of p21CIP1 also resulted in potentiation of apoptosis in cells exposed to FP as well as the combination of SAHA + FP (Figure 7c). Collectively, these findings are consistent with the concept that interference with p21CIP1 induction contributes, at least in part, to enhanced apoptosis observed in leukemic cells exposed to the SAHA/FP combination.
SAHA/FP-mediated lethality proceeds independently of PKC activation and in the presence of a free radical scavenger
To assess the possible role of reactive oxygen species in SAHA/FP-mediated lethality, cells were exposed to these agents in the presence or absence of L-N-acetylcysteine (LNAC), a free radical scavenger that opposes cell death induced by diverse stimuli, including the combination of bryostatin 1 and the proteasome inhibitor lactacystin.22 As shown in Figure 8a, 5 mM LNAC effectively prevented cell death induced by a 24 h exposure to 10 nM bryostatin 1 and 150 nM lactacystin, as we have previously reported,22 but failed to attenuate SAHA/FP-mediated lethality. Similarly, LNAC reduced the loss of ΔΨm triggered by lactacystin and bryostatin 1, but not that induced by SAHA/FP (Figure 8b). Similar results were obtained when 20 mM LNAC was employed (data not shown). In addition, the PKC inhibitor GFX (1 μM) also failed to attenuate SAHA/FP-induced apoptosis. In separate studies, concentrations of GFX ranging from 1 to 10 μM exerted similar effects, and blocked PMA-induced induction of p21CIP1 (data not shown). These findings suggest that the lethal effects of SAHA/FP in human leukemia cells proceed independently of PKC activation and may involve factors other than or in addition to ROS generation.
Ectopic expression of CrmA or dominant-negative caspase-8 partially attenuate SAHA/FP-mediated lethality
Because cross-talk is known to exist between the intrinsic, mitochondrial, and the extrinsic, receptor-related pathway,28 the effects of a 24 h exposure to SAHA/FP were examined in U937 cells ectopically expressing either dominant-negative caspase-8 or CrmA (Figure 9). Parallel studies were performed utilizing TNFα + cycloheximide as control for receptor-mediated apoptosis. As shown in Figure 9a, ectopic expression of CrmA and particularly DN caspase-8 blocked TNF/cycloheximide-induced apoptosis. In addition, SAHA/FP-induced apoptosis was also significantly, albeit partially, reduced in these mutant cell lines. Parallel results were obtained when procaspase-8 and Bid degradation were monitored (Figure 9b). Such findings indicate that activation of the extrinsic, caspase-8-associated pathway contributes to the enhanced apoptosis observed in SAHA/FP-treated cells. However, when clonogenic survival was monitored, neither ectopic expression of CrmA or DN-caspase-8 significantly protected clonogenic cells from the lethal effects of the SAHA/FP combination (Figure 9c). This finding suggests that interference with the extrinsic pathway, despite attenuating apotosis, does not prevent clonogenic cells exposed to SAHA/FP from undergoing a reproductive cell death.
Co-administration of TNF soluble receptor fails to block SAHA/FP-mediated lethality
In view of the ability of ectopic expression of DN-caspase 8 or CrmA to provide partial protection against SAHA/FP-mediated apoptosis, an attempt was made to determine whether this phenomenon might involve release of TNFα. To this end, cells were exposed to SAHA/FP or TNF/CHX in the presence or absence of TNF-soluble receptor (TNFSR), and apoptosis monitored by annexin V/PI staining via flow cytometry (Figure 10). Whereas addition of TNFSR essentially abrogated the lethal effects of TNF/CHX (Figure 10, last two panels), it provided no protection to cells exposed to SAHA in combination with FP (Figure 10, second and third panels from the left). Together, these findings are most consistent with the notion that engagement of procaspase-8 and the extrinsic pathway by FP/SAHA occurs secondary to activation of the intrinsic pathway, rather than through initiation of TNF release. However, the possibility that other components of this pathway (ie FasL) might be involved cannot be excluded.
Ectopic expression of Bcl-2 is minimally effective in protecting U937 cells from SAHA/FP-mediated cytochrome c release, apoptosis, and loss of clonogenic potential
To determine whether and to what extent Bcl-2 might protect cells from SAHA/FP-induced apoptosis, studies were carried out in U937 cells transfected with a plasmid encoding full-length Bcl-2 (U937/Bcl-2). These cells exhibit approximately a 300% increase in Bcl-2 expression relative to empty vector controls (U937/pCEP4), and have previously been shown to be highly resistant to cytotoxic drugs such as ara-C23 and paclitaxel.29 As shown in Figure 11a, ectopic expression of Bcl-2 was minimally effective in blocking apoptosis and loss of ΔΨm induced by a 24 h exposure to 100 nM FP and 1.0 μM SAHA. In contrast, ectopic expression of Bcl-2 was clearly effective in reducing apoptosis induced by ara-C (Figure 11a and b), in accordance with our earlier results.23 Moreover, increased expression of Bcl-2 was also relatively ineffective in preventing cytochrome c release by the SAHA/FP drug combination (Figure 11c). These observations are consistent with previous reports indicating that both FP alone30 as well as the combination of FP and PMA19 trigger apoptosis through a Bcl-2-independent mechanism. In separate studies, cell cycle changes in Bcl-2-overexpressing cells exposed to SAHA/FP did not differ significantly from those observed in empty-vector control cells (data not shown). Finally, in view of evidence that ectopic expression of Bcl-2 may be unable to protect clonogenic cells from certain forms of drug-induced lethality,31 colony-forming assays were carried out following exposure of U937/pCEP4 and U937/Bcl-2 to 100 nM FP and 1.0 μM SAHA for 24 h (Figure 11d). These studies revealed that combining SAHA with FP resulted in a significant (eg ∼1 log) loss of clonogenic potential relative to FP alone. Furthermore, ectopic expression of Bcl-2 afforded clonogenic cells no protection from the lethal effects of FP alone, as previously reported,32 or the combination of FP + SAHA. The latter results are similar to those noted in cells ectopically expressing CrmA or DN-caspase-8, in which restoration of clonogenic growth also did not occur (Figure 9c).
The present results indicate that combined exposure of human leukemia cells, including those of both myeloid and lymphoid lineage, to the histone deacetylase inhibitor SAHA and the CDK inhibitor FP, results in a marked increase in mitochondrial damage, caspase activation, and apoptosis. Such findings are concordant with those previously observed in leukemic cells treated with FP in combination with the PKC activator PMA,19 an agent known to be a potent stimulus for leukemic cell differentiation.33 Previous studies have demonstrated that histone deacetylase inhibitors such as sodium or phenylbutyrate effectively induce human leukemic cell maturation,34 and SAHA has been shown to induce differentiation in MEL cells.1 Given accumulating evidence that differentiation and apoptosis represent alternative and in many cases mutually exclusive events,35 it is tempting to speculate that FP disrupts the ‘normal’ maturation program of leukemic cells exposed to various differentiation inducers, thereby triggering an apoptotic response. The finding that FP antagonized SAHA-mediated maturation, reflected by diminished CD11b expression (Figure 6b), is consistent with this notion. However, in view of the ability of SAHA to induce apoptosis in human leukemia cells, particularly when administered above threshold concentrations,9 the alternative possibility that SAHA/FP-mediated cell death prevents cells from engaging a maturation program cannot be excluded.
The ability of FP to block induction of p21CIP1 in SAHA-treated cells in a caspase-independent manner in many respects resembles its capacity to prevent PMA-induced p21CIP1 up-regulation.19 Induction of p21CIP1 plays a key role in the differentiation process, and has been identified as an important determinant of whether leukemic cells undergo maturation or apoptosis in response to various stimuli, including PKC activators.36 Thus, disruption of p21CIP1 induction (ie in cells expressing stable antisense constructs) promotes apoptosis in leukemic cells exposed to diverse agents, including PMA,37 vitamin D3,38 and sodium butryate.27 In this regard, there is evidence that p21CIP1 exerts anti-apoptotic actions that are distinct from its cell cycle inhibitory properties.39 For example, cytoplasmic localization of p21CIP1 has been shown to attenuate apoptosis by binding to and inhibiting caspase-3.40 Because HDIs such as butyrate41 and SAHA9 are potent inducers of p21CIP1, the possibility arises that this CDKI acts to limit the extent of apoptosis triggered by such agents. If this is the case, dysregulation of p21CIP1 (ie by FP) would be expected to lower the apoptotic threshold, a concept supported by the observation that cells exhibiting dysregulated p21CIP1 expression were more sensitive to apoptosis induced by SAHA alone as well as the SAHA/FP combination. The mechanism by which FP might exert such an action is uncertain, but could be related to its recently described capacity to form DNA duplexes42 and/or inhibit transcriptional activity through inhibition of CDK9.43 Additional studies will be required to resolve these issues, and to determine what, if any, role disruption of p21CIP1 induction plays in the pro-apoptotic and anti-differentiation actions of FP.
The present findings suggest that while leukemic cell apoptosis following exposure to SAHA + FP stems from potentiation of mitochondrial injury, they also raise the possibility that engagement of the extrinsic, caspase-8-dependent pathway participates in this response. Although initial studies suggested that FP triggered apoptosis through a Bcl-2-independent, caspase-8-mediated mechanism, at least in some neoplastic cells,30 more recent studies indicate that FP is a potent inducer of mitochondrial damage (ie cytochrome c release) in human leukemia cells.32 Similarly, HDIs such as butyrate have been shown to promote mitochondrial damage, including release of cytochrome c, in neoplastic cells.44 Although SAHA, administered alone at concentrations lower than 1.5 μM, failed to trigger significant mitochondrial damage, a pronounced increase in cytochrome c release was noted when it was combined with FP. Such findings raise the possibility that these agents may interact by lowering the threshold for mitochondrial injury. Moreover, the observation that caspase inhibition (eg by BOC-D-fmk) failed to block FP/SAHA-mediated cytochrome c release indicates that the latter event lies upstream of activation of the caspase cascade. However, the finding that ectopic expression dominant-negative caspase-8 or CrmA diminished, although only partially, FP/SAHA-induced apoptosis implicates the extrinsic, caspase-8-dependent pathway in cell death associated with this drug combination. While caspase-8 can function as an initiator caspase in receptor-mediated cell death, it can also serve as an executioner caspase in cells that have sustained mitochondrial injury (eg in response to certain chemotherapeutic agents).28 Under such circumstances, cytochrome c released into the cytoplasm, in conjunction with apaf-1 and dATP, promotes activation of procaspase-9.45 This leads in turn to activation of procaspase-3 followed by cleavage of the BH3-only domain Bcl-2 family member Bid, which undergoes mitochondrial translocation and promotes further cytochrome c redistribution.46 The ability of DN-caspase-8 and CrmA to attenuate FP/SAHA-mediated lethality is consistent with the notion that caspase-8 acts to amplify the primary mitochondrial injury triggered by these agents. On the other hand, the finding that TNF-soluble receptor failed to modify FP/SAHA lethality argues against the possibility that these agents act upstream (ie at the level of TNF release) in the extrinsic apoptotic cascade.
The observation that treatment of U937 cells with FP/SAHA resulted in a pronounced increase in Bid cleavage is concordant with results of a recent study in which SAHA-mediated lethality in CCRF-CEM cells was associated with Bid activation and cytochrome c release.47 However, in the latter study, CrmA failed to protect CCRF-CEM cells from SAHA lethality, whereas in the present study, partial protection from SAHA/FP-mediated apoptosis was noted. In addition, while L-N-acetylcysteine protected CCRF-CEM cells from SAHA-associated cytotoxic effects,47 implicating ROS generation in SAHA lethality, this free radical scavenger failed to block FP/SAHA-mediated apoptosis in U937 cells. The disparities between these findings may reflect cell type-specific differences, or, alternatively, the possibility that SAHA- and SAHA/FP-mediated lethality proceeds along separate, albeit related, apoptotic pathways.
The relative inability of ectopic expression of Bcl-2 to protect leukemic cells from FP/SAHA-mediated apoptosis is noteworthy, and is consistent with previous findings that FP alone,30,32 or the combination of FP and PMA,19 induce cell death in a Bcl-2-independent manner. Overexpression of Bcl-2 was also ineffective in restoring clonogenic survival to leukemic cells exposed to the FP/SAHA combination. In this regard, several previous studies have shown that ectopic expression of Bcl-2 may delay, but not ultimately prevent, apoptosis triggered by various cytotoxic agents, resulting in a continuing loss of self-renewal capacity.31 The inability of Bcl-2 overexpression to restore clonogenicity to FP/SAHA-treated cells may therefore reflect failure to prevent apoptosis, selective toxicity to self-renewing cells, or a combination of these factors. It is also noteworthy that while ectopic expression of CrmA or DN-caspase 8 did partially attenuate FP/SAHA-related apoptosis, clonogenicity was not restored. Such a finding is consistent with recent reports that in the presence of caspase inhibition, cells that have sustained significant mitochondrial injury may die an alternative, non-apoptotic form of cell death.48,49
In summary, the present studies indicate that co-administration of the HDI SAHA with the CDK inhibitor FP results in a highly synergistic induction of apoptosis in human leukemia cells. Moreover, this phenomenon is associated with the caspase-independent release of cytochrome c, the caspase-dependent loss of ΔΨm, Bcl-2 cleavage, pRb degradation, and antagonism of SAHA-mediated p21CIP1 upregulation and differentiation. Co-administration of SAHA and FP also effectively induces apoptosis in and inhibits the clonogenic survival of leukemic cells ectopically expressing Bcl-2. Such events mimic those previously observed in leukemic cells exposed to FP in conjunction with the potent differentiation-inducing agent and PKC activator PMA,19 thereby demonstrating that this interaction can be now be extended to other classes of maturation inducers including HDIs. In this context, we have recently observed synergistic antileukemic interactions between FP and the HDI sodium butyrate, which is approximately 1000-fold less potent on a molar basis than SAHA.50 It may be significant that FP also blocked p21CIP1 induction by butryate in U937 cells. Whether FP-mediated disruption of cell cycle-related events (eg induction of p21CIP1) and/or direct potentiation of mitochondrial injury represent primary mechanisms responsible for such interactions remains to be determined. In any case, given accelerating interest in the development of HDIs, including SAHA, as antineoplastic agents,10 and the feasibility of achieving plasma FP concentrations in humans that significantly exceed those employed in the present studies,17 further development of an antileukemic strategy combining SAHA with the CDK inhibitor FP appears warranted. Accordingly, such studies are currently underway.
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This work was supported by awards CA 63753 and CA 83705 from the NIH, and award 6630–01 from the Leukemia and Lymphoma Society of America.
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Almenara, J., Rosato, R. & Grant, S. Synergistic induction of mitochondrial damage and apoptosis in human leukemia cells by flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Leukemia 16, 1331–1343 (2002). https://doi.org/10.1038/sj.leu.2402535
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