Affiliations of authors: Department of Molecular Virology, Immunology, and Medical Genetics (SR, MAC), Medical Scientist Program (SR, BWB, AGF, MAC), Department of Internal Medicine (RAB, SV, DB, JC, KKC, CFE, MRG, MAC), Division of Hematology/Oncology (RAB, JC, CFE, MRG, MAC), Integrated Biomedical Graduate Program (BWB, AGF), College of Pharmacy (Chang-Shi Chen, JJX, KKC, Ching-Shih Chen), Comprehensive Cancer Center (JJX, KKC, CFE, Ching-Shih Chen, MAC), Experimental Therapeutics Program (KKC, MP, MRG), Department of Molecular and Cellular Biochemistry (MP), Division of Epidemiology and Biometrics (AKF), The Ohio State University, Columbus
Correspondence to: Michael A. Caligiuri, MD, A458 Starling Loving Hall, 320 W. 10th Ave., Columbus, OH 43210 (e-mail: caligiuri-1{at}medctr.osu.edu)
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ABSTRACT |
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INTRODUCTION |
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Immune-compromised individuals are at increased risk for developing aggressive EBV-associated lymphoproliferative disorders after primary EBV infection or reactivation of a preexisting latent EBV infection. EBV-associated malignancies are characterized by specific patterns of latent EBV gene expression. Burkitt lymphomas display a Lat-I expression profile, Hodgkin disease and nasopharyngeal carcinoma express a Lat-II expression profile, and EBV-associated lymphoproliferative disorders in individuals with an immune deficiency generally display a Lat-III expression profile (1). Because most EBV-positive malignancies are associated with high mortality, new treatment strategies that specifically target EBV-transformed cells are needed (1,2).
In the past 10 years, a novel class of anticancer drugs that inhibit histone deacetylase enzymes has been developed. Histone deacetylases participate in the remodeling of chromatin structure by removing acetyl groups from lysine residues in the amino-terminal region of histones. Histone deacetylation stabilizes chromatin structure and represses transcription. Inhibition of histone deacetylases allows multisubunit complexes with histone acetyltransferase activity to covalently modify core histones that in turn alter chromatin conformation, making promoter regions more accessible to transcription factors and thus more permissive to transcriptional activation. Differentiation, apoptosis, and/or cell cycle arrest have been induced in several cancer cell lines in vitro by histone deacetylase inhibitors (3). Depsipeptide is a histone deacetylase inhibitor that has shown promising anticancer activity and been safely administered to patients with refractory neoplasms (4) and to patients with cutaneous T-cell lymphomas (5).
To our knowledge, the effect of histone deacetylase inhibition on the survival of a xenograft mouse model of human B-cell lymphoma has not been explored. The purpose of this study was to evaluate the effects of depsipeptide, a histone deacetylase inhibitor, on EBV-positive lymphoblastoid and Burkitt lymphoma cell lines in a xenograft mouse model and to explore its mechanism of action.
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MATERIALS AND METHODS |
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Human blood samples were procured under an Institutional Review Boardapproved protocol at The Ohio State University Hospitals. We used three different human EBV-positive LCLs, two of which (PA-LCL and RM-LCL) had been developed in vitro from the peripheral blood of two patients diagnosed with a post-transplant lymphoproliferative disorder, and the third (C7M3) had been derived from a severe combined immune-deficient (SCID) mouse engrafted with human peripheral blood lymphocytes from a healthy EBV-positive donor (6). EBV-negative Burkitt lymphoma cell lines (BL-41 and Ramos) and EBV-positive Burkitt lymphoma cell lines (Raji, Daudi, and Jijoye) were obtained from the American Type Culture Collection (Manassas, VA). All cell lines were cultured in standard growth medium containing RPMI 1640 medium supplemented with antibiotics (penicillin at 100 U/mL and streptomycin at 100 µg/mL) and 10% fetal bovine serum (Invitrogen, Rockville, MD). All cell lines were mycoplasma free and were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Viability and cell counts were determined by trypan blue dye exclusion.
For cell cycle synchronization experiments, cells were synchronized in G1 phase by an overnight exposure (10 hours) to aphidicolin at 5 µg/mL (Sigma, St. Louis, MO). The cell cycle distribution was then confirmed via flow cytometry to assess the DNA content of cells after propidium iodide staining as described previously (11). After this treatment, 80% of cells were in G1 phase.
Treatment With Histone Deacetylase Inhibitors
Stock solutions of 3 mM trichostatin A or 3 M sodium butyrate (Sigma) were prepared in dimethyl sulfoxide (DMSO). Depsipeptide (5 µM) (Fujisawa Pharmaceutical) was dissolved in acetonitrile. Cells were incubated for 4 hours with vehicle control (DMSO or acetonitrile), trichostatin A (3, 3 x 101, or 3 x 102 µM), sodium butyrate (3 x 103, 3 x 104, or 3 x 105 µM), or depsipeptide (1 x 102, 5 x 102, 1 x 101, or 5 x 101 µM) for 4 hours. Cells were then washed twice with phosphate-buffered saline (PBS) and plated in standard growth medium (2 mL of RPMI 1640 medium with 10% fetal bovine serum and antibiotics per well, in triplicate wells of a 24-well plate; Falcon). For apoptosis and proliferation assays, 0.5 x 106 to 1.0 x 106 cells were harvested at 0, 24, 48, or 72 hours after exposure to histone deacetylase inhibitors or the vehicle control.
Animal Model
Six- to eight-week-old female SCID mice were purchased from Taconic Farms (Germantown, NY) and were maintained in pathogen-free, isolated cages. To improve tumor engraftment, all animals were depleted of murine natural killer cells with a single intraperitoneal injection of 0.2 mg of rat antimouse interleukin 2 receptor monoclonal antibodies (TM
1) 2 days before engraftment with human tumor cell lines, as described previously (7). To ensure the consistency of cellular preparations for all in vivo experiments, we first cultured each cell line until a total of 10 x 109 cells were obtained. Frozen stocks (3 x 107 cells per vial) were then prepared in DMSO freezing medium (Sigma), frozen under controlled rate at 86 °C (using Control rate freezing container; Nalgene), and stored in a vapor-phase liquid nitrogen environment (200 °C). Before in vivo passage, cryopreserved cells were thawed, cultured for 10 days, and then resuspended in PBS at room temperature for injection. We performed celldose titration trials to determine an optimal dose of cells that would lead to equivalent fatal tumor burdens in all mice (data not shown) and found that the optimal dose for both C7M3 EBV-positive LCLs and Raji EBV-positive Burkitt lymphoma tumor cells was 10 x 106 cells. Without intervention, mice carrying these tumor cells at the optimal dose have a mean survival time of 20 days.
For in vivo treatment, depsipeptide was suspended with sonication at 0.96 mg/mL in a solution of 10% HCO-60 and PBS at room temperature. HCO-60 is an emulsifying agent (Lipo Chemicals, Paterson, NJ) designed for slow drug release. One milliliter of this depsipeptide solution was diluted with 0.5 mL of 10% ethanol40% propylene glycol in PBS and stored at 4 °C (final concentrations = depsipeptide at 0.64 mg/mL, 66% HCO-60, 3.3% ethanol, and 13.3% propylene glycol). Each day, a depsipeptide working stock was prepared by diluting 0.492 mL of the 0.64-mg/mL solution with 1.6 mL of sterile PBS (final depsipeptide concentration = 0.15 mg/mL).
For toxicity studies, mice were injected intraperitoneally each day with depsipeptide at 3, 5, or 10 mg/m2 of body surface area (n = 3 mice per group). For pharmacokinetic studies, plasma levels of depsipeptide after a single injection of drug (5 mg/m2, n = 3 mice per time point) were determined with a sensitive electrospray liquid chromatographytandem mass spectroscopy assay, as previously described (4,8). For therapeutic studies, mice with tumors (groups of 10 mice) were injected intraperitoneally with 0.2 mL of depsipeptide (0.03 mg per mouse = 5 mg/m2) or with 0.2 mL of vehicle control on days 2, 4, 7, and 10 after tumor inoculation. Animals were monitored daily for signs of tumor burden, including weight loss, ruffled coat, and distended abdomen. When mice showed signs of tumor burden, they were killed with a lethal dose of anesthesia (ketaminexylazine) followed by cervical dislocation. All animal research was reviewed and approved by the University Laboratory Animal Resources at The Ohio State University.
Flow Cytometric Analysis
After treatment with a histone deacetylase inhibitor, 0.5 x 106 to 1 x 106 cells (C7M3, PA, RM, Raji, Daudi, Jijoye, BL41, and Ramos), were harvested, washed, and assayed for apoptosis by dual staining with Annexin Vcoupled fluorescein isothiocyanate (FITC) and propidium iodide (BD Pharmingen, San Diego, CA). Cells were washed in ice-cold PBS and resuspended in Annexin V binding buffer containing 5 µL of Annexin VFITC and 1.5 µL of propidium iodide (50 µg/mL). After a 10-minute incubation at room temperature in the dark, samples were analyzed on a FACScalibur flow cytometer (Becton Dickinson, San Diego, CA). Results were interpreted as follows: Annexin Vpositive and propidium iodidenegative events were considered apoptotic events, Annexin Vpositive and propidium iodidepositive events were considered nonviable (apoptotic or necrotic), and Annexin Vnegative and propidium iodidenegative events were considered viable (9).
Intracellular staining was performed with monoclonal antibodies specific for active caspase 3 (FITC-conjugated from BD Pharmingen), LMP1 (phycoerythrin-conjugated from BD Pharmingen), BCL2 (detected by secondary staining with allophycocyanin-conjugated goat antimouse monoclonal antibody; Molecular Probes, Eugene, OR), and isotype control monoclonal antibodies (BD Pharmingen). Intracellular staining was performed with an intracellular flow cytometry kit (BD Pharmingen) as described previously (10). In brief, cells were simultaneously fixed, permeabilized with the Cytofix/CytoPerm reagent, and then washed in Perm/Wash buffer. Nonspecific binding of monoclonal antibodies was blocked by preincubating samples with unconjugated mouse immunoglobulin G (Sigma). Flow cytometric data were analyzed with CellQuest (Becton-Dickinson) and graphed with WinMDI (Scripps Research Institute). Data are expressed as mean fluorescent intensity, representing populations gated on the specific parameters indicated. For determination of cell cycle and DNA fragmentation status, we measured DNA content with propidium iodide staining of permeabilized cells as described previously (11). Cells were stained with 20 µL of propidium iodide (50 µg/mL) and 5 µL of RNase (10 mg/mL). Cells were protected from light by wrapping tubes in foil and stored for 2 hours at 4 °C before sample collection during flow cytometry. Cell cycle data were analyzed with ModFit (Verity Software House, Topsham, ME).
Proliferation Assay With [3H]Thymidine Incorporation
After a 4-hour treatment with vehicle or 10, 50, 100, or 500 nM depsipeptide, cells in 100 µL of standard growth medium (RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin) were cultured in a 96-well U-bottom plate at 25 000 cells per well (six wells per treatment) for 60 hours. Cells in each well were incubated with 1 µCi of [3H]thymidine (NEN Life Sciences, Boston, MA) for an additional 12 hours at 37 °C, 5% CO2 and 95% HEPA (0.3-µm pore size)filtered air. Mitomycin C blocks DNA replication, resulting in G1/G0 arrest, and was used as a control for growth inhibition. Cells were harvested onto nitrocellulose filters, and [3H]thymidine incorporation was measured with a Beckman Coulter liquid scintillation counter. The proliferation index was expressed as the mean counts per minute in the six replicate wells (with 95% confidence intervals [CIs]).
Effect of Caspase Inhibition on Depsipeptide-Mediated Apoptosis
The broad-spectrum caspase inhibitor z-VAD-fmk (Enzyme Product Systems, Livermore, CA) was dissolved in DMSO for stock solution (15 mM) and further diluted in standard growth medium at concentrations used in experiments. Cell lines were incubated with DMSO (vehicle control, diluted in complete cell culture medium) or with 10150 µM z-VAD-fmk for 1 hour at 37 °C. Cells were then treated for 4 hours with acetonitrile vehicle (diluted 1 : 10 [vol/vol] in standard culture medium), 100 nM or 500 nM depsipeptide, or the agonistic anti-Fas (CD95) monoclonal antibody 7c11, as described previously (12), at 1 µg/mL. Cells were washed twice with sterile PBS, plated in growth medium containing the DMSO control or z-VAD-fmk concentrations in triplicate wells in a 24-well plate, and harvested after 24, 48, or 72 hours of culture. DNA content was analyzed by flow cytometry and agarose gel electrophoresis as described previously (13,14).
Western Blot Analysis
Cleaved (active) caspase 3 was measured by western blot analysis as previously described (15). Treated cells were collected, washed with PBS, resuspended in sodium dodecyl sulfate (SDS) gel-loading buffer (100 mM TrisHCl [pH 6.8], 4% [wt/vol] SDS, 0.2% bromophenol blue, 20% [vol/vol] glycerol, and 200 mM dithiothreitol), sonicated with an ultrasonic sonicator for 5 seconds, and boiled for 5 minutes. After brief centrifugation (10 000g for 2 minutes at 4 °C), equivalent amounts 10 µg of soluble protein, as determined by the Bradford method, were resolved by SDSpolyacrylamide gel electrophoresis in 10% minigels and transferred to nitrocellulose membranes with the use of a semidry transfer cell (Bio-Rad). The membranes were washed twice with Tris-buffered saline (TBS; 0.3% Tris, 0.8% NaCl, and 0.02% KCl) containing 0.05% Tween 20 (TBST) and then incubated with TBS containing 5% nonfat dry milk for 60 minutes to block nonspecific antibody binding. Each membrane was then incubated at 4 °C for 12 hours with a primary antibody specific for caspase 3 (Santa Cruz Biotechnology, Santa Cruz, CA), which was diluted 1 : 1000 in TBS containing 1% nonfat dry milk. The membranes were washed twice with TBST and then incubated at room temperature for 1 hour with a horseradish peroxidaseconjugated goat antirabbit immunoglobulin G diluted 1 : 5000 in TBS containing 1% nonfat dry milk. The membranes were washed twice with TBST, and bound antibody was visualized by enhanced chemiluminescence (ECL) using ECL western blotting detection reagents (Amersham Pharmacia Biotech, Little Chalfont, U.K.).
For evaluation of histone acetylation, we purified histone protein fractions from LCL and Burkitt lymphoma cell lines (C7M3, PA, RM-LCL, Raji, Daudi, Jijoye, and BL41), and western blot analysis was performed as previously described (16). In brief, duplicate purified histone protein preparations were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. One of the gels was stained with Coomassie blue to compare total protein levels, and the other gel was blotted onto a nitrocellulose membrane for western blot analysis. Western blots were probed with antibodies that recognize acetylated lysine residues at positions 5, 8, 12, and 16 within the amino-terminal tail of histone H4 (all monoclonal antibodies from Serotec, Raleigh, NC). Western blots were visualized by ECL as described in the manufacturers instructions (Amersham, Piscataway, NJ).
Nuclear Factor B Detection and Inhibition
The p65 subunit of nuclear factor B (NF-
B) was measured in the nuclear protein fraction of depsipeptide-treated cells and untreated control cells by use of an NF-
B Trans-Am kit (Active Motif, Carlsbad, CA). Cells were harvested after a 4-hour treatment with depsipeptide. For depsipeptide-treated EBV-positive LCLs (C7M3, PA, and RM), viable cells were isolated by centrifugation of suspended cells over a 5-mL FicollHypaque (Sigma) layer for 30 minutes (25 °C); cell viability was verified by trypan blue dye exclusion. Nuclear extracts were prepared from 5 x 106 to 10 x 106 cells with the NE-PER extraction kit, as described by the manufacturer (Pierce Biotechnology, Rockford, IL). Nuclear proteins (1 µg per well, measured with a standard Bradford protein assay) were loaded into quadruplicate wells of a Trans-Am 96-well plate and incubated for 1 hour at room temperature. These plates were coated by the manufacturer with NF-
B consensus oligonucleotides that serve as immobilized substrates to which active NF-
B complexes bind. Plates were washed and probed with antip65 subunit primary monoclonal antibodies and then with horseradish peroxidaseconjugated secondary monoclonal antibodies. After reactions with the horseradish peroxidase substrate and then with stopping reagents provided by the kit, absorbance at 450 nm was read with a spectrophotometer (Perkin-Elmer). Tumor necrosis factor
stimulated HeLa cell extracts were used as positive controls, as described in the manufacturers recommendations. To determine specificity, we added wild-type and mutant NF-
B consensus oligonucleotides to HeLa extracts as additional negative and positive controls, respectively. Relative NF-
B activity was expressed as the average absorbance units per microgram of protein.
PS-1145 is a small molecule (PS-1145 dihydrochloride salt, molecular weight = 395.67; a gift from Millennium Pharmaceuticals) that specifically blocks NF-B activation by inhibiting I
B kinasemediated phosphorylation of I
B
, the key negative regulator of NF-
B activity (17). This inhibitor allows I
B
to evade proteasomal degradation and form stable heterodimers and/or homodimers with NF-
B subunits, preventing its translocation to the nucleus. Stock solutions of PS-1145 were made by dissolving lyophilized drug in DMSO to a final concentration of 10 mM. For experiments involving inhibition of NF-
B, cells were pretreated with 20 µM PS-1145 for 1 hour at 37 °C and then treated with depsipeptide or vehicle control for 4 hours at 37 °C. After treatment, cells were washed free of depsipeptide and PS-1145 twice with sterile PBS and then plated in growth medium containing 20 µM PS-1145 or control.
Statistical Analysis
The mean survival time, 95% confidence interval, and median survival time were calculated for the groups treated with depsipeptide and vehicle control by use of the KaplanMeier estimate (18). The survival times of the groups were compared with the log-rank test (18). This test was performed for each animal model (C7M3 and Raji). Data sets produced in vitro were analyzed with two-sample Students t tests with unequal variances. All statistical tests were two-sided.
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RESULTS |
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To study the effects of depsipeptide in SCID mice, we first performed toxicity studies to determine the maximum tolerated dose and optimal treatment schedule. We evaluated daily injections of depsipeptide at 3, 5, and 10 mg/m2 and found substantial toxicity after a single dose at 10 mg/m2 or after three doses at 5 mg/m2. Rest periods of 23 days between doses at 5 mg/m2 reduced the toxicity substantially; thus, we defined 5 mg/m2 every other day to every third day as the maximum tolerated dose and treatment schedule. We next performed pharmacokinetic studies to determine plasma concentrations of depsipeptide achieved. After a single injection of depsipeptide at the maximum tolerated dose (5 mg/m2), the level of biologically active drug in plasma peaked at 15 minutes at 34 nM (95% CI = 0 to 70; n = 3 mice per sampling time) and then declined rapidly within 4 hours (Fig. 1, A).
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In humans, plasma concentrations of 4501050 nM depsipeptide can be safely achieved over the course of a 4-hour intravenous infusion (4). Consequently, in our in vitro studies, we tested a biologically relevant dose range (50500 nM depsipeptide) and exposure time (4 hours) that were based on the drug levels measured in SCID mice (Fig. 1, A) and the higher doses achieved in human subjects. Apoptosis was induced in 35%64% of cells in a representative culture of depsipeptide-treated EBV-positive LCLs (PA-LCL; compared with 3%6% of cells in a culture of vehicle-treated EBV-positive LCLs) after a single 4-hour treatment with 500 nM depsipeptide (Fig. 2, A). Similar results were obtained with C7M3 LCL and RM LCL. Depsipeptide induced apoptosis in EBV-positive LCLs in a dose-dependent fashion beginning at 10 nM in the C7M3, PA, and RM LCLs (data not shown). In contrast, treatment of EBV-positive or EBV-negative Burkitt lymphoma cell cultures with 500 nM depsipeptide resulted in a marginally higher percentage (14% and 12%, respectively) of apoptotic cells compared with that (3% and 6%, respectively) in vehicle-treated control cultures (Fig. 2, B and C).
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Induction of Apoptosis in EBV-Positive LCLs with Depsipeptide and Other Histone Deacetylase Inhibitors
To compare the proapoptotic activity of the depsipeptide with other compounds with histone deacetylase inhibitor activity, we performed dose-escalation experiments with trichostatin A and butyrate. Dose-escalation experiments with a 4-hour exposure demonstrated that trichostatin A was cytotoxic at concentrations 600-fold greater than depsipeptide and that butyrate was cytotoxic at concentrations 600 000-fold greater than depsipeptide (Fig. 3). In contrast to depsipeptide, however, both compounds were equally cytotoxic to cell lines with Lat-I (Raji) and Lat-III (C7M3, PA, and RM) viral gene expression profiles (data not shown).
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A major pathway leading to apoptosis involves activation of multiple caspase proteins that converge on the cleavage and subsequent activation of caspase 3, which in turn triggers other apoptotic cascades. Treatment of the three EBV-positive LCLs (C7M3, PA, and RM) with depsipeptide induced statistically significantly more active caspase 3 after 48 hours than that in corresponding control cultures treated with vehicle (Fig. 4, A; P<.001). The percentage of cells staining positive for active caspase 3 (Fig. 4, A, lower panel) was associated with levels of apoptosis as determined by Annexin V and propidium iodide staining (Fig. 2, A) and by nuclear DNA staining (Fig. 4, B, right panel). Cells in which active caspase 3 was not detected also had a normal DNA content, with the proportions of cells in G1, S, and G2/M phases expected for proliferating cells (Fig. 4, B, left panel). Inhibition of caspase 3 with the broad-spectrum caspase inhibitor zVAD-fmk before treatment with depsipeptide partially reduced depsipeptide-induced apoptosis from 90% of cells in apoptosis (Fig. 5, A, no zVAD-fmk treatment) to 63% (Fig. 5, B, addition of zVAD-fmk). Western blot analysis for the active form of caspase 3 demonstrated that the broad-spectrum caspase inhibitor zVAD-fmk completely inhibited caspase 3 cleavage (i.e., activation) in EBV-positive LCLs treated with anti-Fas or depsipeptide (Fig. 5, C). These results demonstrate that depsipeptide-induced apoptosis of EBV-positive LCLs occurs via mechanisms that are caspase dependent and mechanisms that are caspase independent.
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We consistently observed a small subset of Lat-III EBVpositive LCLs that remained viable despite treatment with high concentrations of depsipeptide. In repeated experiments, viability was not dependent on phase of the cell cycle (data not shown). Tumor cells resistant to depsipeptide-mediated apoptosis could be clearly identified by flow cytometry as a population that was negative for Annexin V binding, propidium iodide binding, and active caspase 3 (Figs. 2 and 4, respectively). Because the LMP1 protein is critical to survival of EBV-positive LCLs (19,20), we used intracellular flow cytometry to compare LMP1 levels in depsipeptide-sensitive and -resistant populations. Forty-eight hours after treatment with depsipeptide, cells were separated by flow cytometry into an active caspase 3positive population or an active caspase 3negative population, and then LMP-1 expression was evaluated in each population (Fig. 6, A). Subpopulations with low and high LMP1 expression were present before depsipeptide treatment. This heterogeneity in LMP1 expression resulted in a mean fluorescent intensity (MFI) of 39.96 U (Fig. 6, B). Forty-eight hours after a 4-hour exposure to depsipeptide, cells with active caspase 3 were predominantly those expressing low levels of LMP1 (MFI = 13.13 U, Fig. 6, C), whereas cells lacking active caspase 3 expressed higher levels of LMP1 (MFI = 61.77 U, Fig. 6, D) (P = .017). The same pattern was true for the antiapoptotic protein BCL2 (Fig. 6, D; P = .032), a downstream target of LMP1 (20). Thus, expression of LMP1 and BCL2 was associated with caspase 3 activation after treatment with depsipeptide. Given the ability of LMP1 to trigger the activation of several cellular survival pathways, the mechanisms by which EBV-positive LCLs survive depsipeptide treatment likely involve one or more downstream targets of LMP1.
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LMP1 protein can also enhance the survival and growth of EBV-transformed lymphoblasts by constitutive activation of the transcription factor NF-B (21). To investigate whether this potential survival mechanism was operating in depsipeptide-resistant cells, we isolated viable cells (LMP1-positive, BCL2-positive, and caspase 3-negative cells) that survived a high-dose (500 nM) depsipeptide treatment and evaluated their NF-
B activity. These depsipeptide-resistant cells exhibited higher levels of NF-
B p65containing dimer than cells treated with vehicle control (absorbance at 450 nm = 1.57 versus 1.13; P<.001) (Fig. 7, A).
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DISCUSSION |
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As with chromatin in the cells nucleus, the EBV episome is organized into a tightly compacted nucleosomal structure that requires multisubunit enzymatic complexes to alter its nucleosomal conformation and allow coordinated gene expression. The regulation of latent and lytic EBV gene expression is complex and requires both covalent (2225) and ATP-dependent (26,27) mechanisms to modulate promoter accessibility and thus the expression of a specific latent (Lat-I, -II, or -III) or lytic gene profile (2830). It is possible that the antiproliferative effect of depsipeptide in EBV-positive LCLs (Fig. 2) involves activation of lytic transcription, because we have observed an induction of intermediate early lytic gene products after exposure of EBV-positive LCLs to depsipeptide (data not shown) (31). Unlike other histone deacetylase inhibitors that we examined (trichostatin A and butyrate), depsipeptide did not have antiproliferative, proapoptotic activity on Burkitt lymphoma cell lines. Although the mechanism for this apparent selective death of Lat-III EBV-positive LCLs is not clear, depsipeptide-mediated histone deacetylase inhibition may derepress Myc target genes, as described previously by Pal et al. (32). This activity could then enhance the proliferation and survival of EBV-positive Burkitt lymphoma cell lines that possess the IgHc-myc translocation, which drives the constitutive expression of Myc.
Alternatively, depsipeptide may be exerting these selective properties through a mechanism that is independent of histone acetylation. Gu et al. (33) reported that p53 DNA binding affinity is regulated by acetylation of lysine residues in the carboxyl terminus of p53. Treatment with a histone deacetylase inhibitor can result in the acetylation of specific lysine residues in p53, leading to the increased stability and transcription of p53-driven proapoptotic target genes (34,35). Because 60%80% of Burkitt lymphoma cell lines have mutations in p53, this may render them more tumorigenic in vivo and possibly resistant to the effects of acetylation mediated by histone deacetylase inhibitor treatment (36).
We observed a 12-fold increase in the level of active caspase 3 after cells were briefly exposed to depsipeptide. Pretreatment of EBV-positive LCLs with z-VAD-fmk resulted in complete inhibition of caspase 3 cleavage, as detected by western blot analysis, but in only partial inhibition of depsipeptide-induced apoptosis, as determined by flow cytometry with Annexin and propidium iodide staining. Susin et al. (37) reported that apoptosis-inducing factor is a mediator of caspase-independent apoptosis. Although pretreatment of EBV-positive LCLs with the broad-spectrum caspase inhibitor z-VAD-fmk partially inhibited depsipeptide-induced cell death, it effectively prevented high-molecular-weight DNA fragmentation, a characteristic feature of apoptosis induced by apoptosis-inducing factor (data not shown). Thus, it appears that the mechanism of depsipeptide-mediated apoptosis in EBV-positive LCLs is independent of caspase 3 and apoptosis-inducing factor (37).
Despite depsipeptides induction of apoptosis, we observed a subset of cells that were resistant to depsipeptide-induced apoptosis in vitro. This heterogeneity may reflect heterogeneous expression of the Lat-IIIassociated viral gene product LMP1 in EBV-positive LCLs (38). LMP1, a member of the tumor necrosis factor receptor family, uses tumor necrosis factor receptorassociated factors to bypass CD40 signaling leading to the constitutive activation of the survival factors BCL2 and NF-B in EBV-transformed lymphoblasts (19,39,40). We observed a similar degree of heterogeneity of LMP1 expression with both untreated and depsipeptide-treated EBV-positive LCLs; thus, it is possible that EBV-positive LCLs that express high levels of LMP1 and the downstream survival mediators NF-
B and BCL2 would selectively survive exposure to depsipeptide.
It is also possible that depsipeptide-mediated histone deacetylase inhibition leads to changes in LMP1 expression via epigenetic mechanisms. Indeed, several reports have described that histone deacetylase and histone acetyltransferase activity regulate the LMP1 promoter (2426,41,42). In addition, Sjoblom-Hallen et al. (43) demonstrated that expression of LMP1 is silenced by a multisubunit complex containing Max, Mad1, mSin3A, and histone deacetylase that localizes to an E-box site present within the LMP1 promoter. EBNA3C, an EBV transcription factor belonging to the Lat-III viral gene profile, recruits histone deacetylases and represses EBNA2-activated transcription, including that at the LMP1 promoter (24,42). By the use of chromatin immunoprecipitation experiments with monoclonal antibodies specific for EBNA2 and acetylated histones, Alazard et al. (41) showed that the EBV-encoded coactivator EBNA2 binds to the LMP1 promoter and that LMP1 promoterassociated histone H3 acetylation is linked with recruitment of EBNA2. Furthermore, Park et al. (38) showed that treatment with nontoxic doses of the histone deacetylase inhibitors butyrate (2.5 x 103 µM) and trichostatin A (0.332 µM) can induce the expression of LMP1 mRNA and protein in EBV-positive Burkitt lymphomas. To address whether depsipeptide could be having the same effect, we isolated the population of EBV-positive LCLs expressing high levels of LMP1 (LMP1high) and the population of EBV-positive LCLs expressing low levels of LMP1 (LMP1l°w) by flow cytometric sorting of distinct populations of LCLs with high or low CD54 surface expression, as described by Park et al. (38). We found that depsipeptide treatment of EBV-positive LCLs with low CD54 expression (also LMP1l°w) enhanced the expression of LMP1 (data not shown). Thus, depsipeptide-mediated inhibition of histone deacetylase appears to simultaneously induce apoptosis and LMP1 expression, leading to the downstream activation of key survival pathways, including the LMP1 targets BCL2 and NF-B (44).
LMP1 signaling increases the expression of BCL2 in EBV-infected B cells (20). We found an association between survival and the expression of LMP1 and BCL2 in depsipeptide-treated cells (Fig. 6). BCL2 can inhibit both caspase-dependent and -independent apoptosis by limiting the permeability of the mitochondrial membrane, thereby preventing the contents of the mitochondrial matrix from reaching the cytosol. In particular, BCL2 can prevent the release and subsequent activation of caspase-dependent proapoptotic factors, including cytochrome c and Bid (45). An earlier study (40) reported that treatment of EBV-positive LCLs with LMP1 antisense RNA decreased BCL2 levels, reduced proliferative capacity, and induced apoptosis under serum-free conditions. Thus, LMP1-mediated induction of BCL2 would lead to resistance of both caspase-dependent and -independent apoptosis after exposure to depsipeptide.
A second element of LMP1 signal transduction that may contribute to depsipeptide resistance is the constitutive activation of the transcription factor NF-B. LMP1 constitutively activates NF-
B in EBV-positive LCLs through tumor necrosis factor receptorassociated factors, which activate NF-
Binducing kinases and I
B kinases to phosphorylate I
B
, thus promoting I
B
degradation by the proteasome (46). I
B
prevents NF-
B dimerization and binding to target sequences within DNA by binding and sequestering NF-
B subunits in the cytoplasm. Viable EBV-positive LCLs that persisted after depsipeptide treatment had higher levels of NF-
B activity than untreated control EBV-positive LCLs (Fig. 7). Thus, surviving cells may have responded to depsipeptide treatment by increasing their NF-
B activity. To further explore the contribution of NF-
B to this apparent intrinsic resistance to depsipeptide, we inhibited LMP1-mediated activation of NF-
B with PS-1145, a specific inhibitor of I
B kinase (17). Pretreatment of cultures with PS-1145 alone had no effect on cell viability, but combined treatment of cultures with PS-1145 and depsipeptide resulted in increased apoptosis compared with cultures treated with depsipeptide alone. Because preexisting NF-
B activity is not affected by PS-1145, there was only partial inhibition of LMP1-driven NF-
B activation. Expression of a dominant negative NF-
B in EBV-positive LCLs has been reported to cause spontaneous apoptosis (47). It is possible that partial inhibition of NF-
B activity lowered the apoptotic threshold of EBV-positive LCLs, lending support to the hypothesis that NF-
B is a resistance factor against depsipeptide-induced apoptosis. Indeed, recent work has identified binding sites for several components of NF-
B complexes within the BCL2 (48) and A1/Bfl1 (49) promoters, suggesting that important survival gene products are downstream targets of NF-
B activity.
With the more frequent use of stem cell and solid organ transplantation and the accelerating worldwide AIDS epidemic, the number of individuals with chronic immune suppression will continue to increase. Thus, it is vital to continue development of novel strategies that effectively target virally associated opportunistic neoplasms in immune-compromised individuals. In an era where drug development has shifted toward molecular targeting, reagents with histone deacetylase inhibitor activity are emerging as attractive compounds to test in clinical trials. Compounds with histone deacetylase inhibitor activity induce growth arrest, differentiation, and apoptosis in various tumor cell lines in vitro and in several preclinical animal models of cancer (3). Although the development of many of these compounds has been limited by their suboptimal pharmacokinetics and high toxicity profiles (50), depsipeptide is a potent histone deacetylase inhibitor that can reach plasma concentrations of 4001000 nM in humans and has a proven safety record in phase I trials (4,5). Although the potential of depsipeptide as a treatment for EBV-associated B-cell malignancies has yet to be explored clinically, the in vivo and in vitro data from this study suggest that use of depsipeptide as single agent, followed by a combination treatment with depsipeptide and PS-1145, would be a reasonable approach to the treatment of EBV-associated B-cell lymphoproliferative disorders.
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Supported by National Institutes of Health grant T32 CA09338 to R. A. Baiocchi and by National Institutes of Health grant P01 CA95426 to C. F. Eisenbeis and M. A. Caligiuri.
We thank Millennium Pharmaceuticals, Inc., for PS-1145 compound. We also thank John Wright from the National Cancer Institute and Fujisawa Pharmaceuticals, Inc., for providing depsipeptide. We thank Donna Bucci, Tamra Brooks, and Karen Feasel for administrative assistance and preparation/submission of the manuscript.
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Manuscript received January 23, 2004; revised July 29, 2004; accepted August 15, 2004.
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