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(The FASEB Journal. 1999;13:1991-2001.)
© 1999 FASEB

Apoptosis induced by the histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts

DAVID BERNHARD, MICHAEL J. AUSSERLECHNER, MARTIN TONKO, MARKUS LÖFFLER, BERND L. HARTMANN, ADAM CSORDAS* and REINHARD KOFLER1

Institute for General and Experimental Pathology, Division of Molecular Pathophysiology; and
* Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Medical School, Innsbruck, Austria, A-6020

1Correspondence: Institute for General and Experimental Pathology, Division of Molecular Pathophysiology, Fritz-Pregl-Straße 3, A-6020 Innsbruck, Austria. E-mail: Reinhard.Kofler{at}uibk.ac.at


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The histone deacetylase inhibitor and potential anti-cancer drug sodium butyrate is a general inducer of growth arrest, differentiation, and in certain cell types, apoptosis. In human CCRF-CEM, acute T lymphoblastic leukemia cells, butyrate, and other histone deacetylase inhibitors caused G2/M cell cycle arrest as well as apoptotic cell death. Forced G0/G1 arrest by tetracycline-regulated expression of transgenic p16/INK4A protected the cells from butyrate-induced cell death without affecting the extent of histone hyperacetylation, suggesting that the latter may be necessary, but not sufficient, for cell death induction. Nuclear apoptosis, but not G2/M arrest, was delayed but not prevented by the tripeptide broad-range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp·fluoromethylketone (zVAD) and, to a lesser extent, by the tetrapeptide `effector caspase' inhibitors benzyloxycarbonyl-Asp-Glu-Val-Asp·fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Glu-Ile-Asp·fluoromethyl-ketone (VEID); however, the viral protein inhibitor of `inducer caspases', crmA, had no effect. Bcl-2 overexpression partially protected stably transfected CCRF-CEM sublines from butyrate-induced apoptosis, but showed no effect on butyrate-induced growth inhibition, further distinguishing these two butyrate effects. c-myc, constitutively expressed in CCRF-CEM cells, was down-regulated by butyrate, but this was not causative for cell death. On the contrary, tetracycline-induced transgenic c-myc sensitized stably transfected CCRF-CEM derivatives to butyrate-induced cell death.—Bernhard, D., Ausserlechner, M. J., Tonko, M., Löffler, M., Hartmann, B. L., Csordas, A., Kofler, R. Apoptosis induced by the histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts.


Key Words: caspase • c-myc • bcl-2 • p16/INK4A • CCRF-CEM


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BUTYRATE IN MILLIMOLAR concentrations has long been known to be an inhibitor of histone deacetylases and to cause reversible G0/G1 growth arrest and induction of differentiation markers in cells from numerous species and tissues (reviewed in refs 1 , 2 ). More recently, butyrate has been recognized to induce apoptosis in certain nontransformed as well as transformed cell types, including colorectal (3 , 4) , breast (5 , 6) , hepatic (7) , and hematopoietic malignant (8 9 10) cell lines. Due to its growth-inhibiting and differentiation-inducing ability, butyrate as well as its analogs with better pharmacodynamic properties were tested, alone or in combination with other anti-cancer drugs, in the therapy of solid tumors (11) and leukemias (12 13 14) .

The physiological and therapeutic effects of butyrate are thought to result primarily from core histone hyperacetylation (15 , 16) , because chemically unrelated histone deacetylation inhibitors like trichostatin A or trapoxin, have been shown in several instances to mimic the effects of butyrate (17) . Histone acetylation, in turn, has long been claimed to influence gene expression, a notion strongly supported by recent reports showing that components of the basal transcription machinery and several sequence-specific transcription factor coactivators have histone acetyl transferase activity, whereas corepressors often recruit histone deacetylases (reviewed in refs 18 19 20 21 ). Thus, histone hyperacetylation is thought to facilitate, and deacetylation to repress, individual gene expression. Nevertheless, global histone hyperacetylation, whether induced genetically or pharmacologically, does not lead to a general increase in gene transcription (20) , and only a limited number of genes are up- or down-regulated by butyrate (summarized in 2 ). The transcriptional regulation of some of these genes explains the compound's biological effects as, for instance, the derepression of the fetal gamma-globin genes in the treatment of ß-hemoglobinopathies (22 , 23) . The genes responsible for inhibition of proliferation and induction of cell differentiation or death by butyrate continue to remain elusive, although some promising candidates have been identified, e.g., the cyclin-dependent kinase inhibitor p21/WAF1 (24 25 26) , c-myc (4 , 10) , and the anti-apoptotic bcl-2 gene (6 , 27) (see Discussion).

In this study, we investigated the effect of butyrate on the human acute T cell leukemia model, CCRF-CEM (28) . The drug induced inhibition of proliferation, accumulation of cells in the G2/M phase of the cell division cycle, and typical apoptotic cell death. Since butyrate and similar drugs represent potential agents for leukemia therapy, we determined several molecular characteristics of this apoptosis pathway. In particular, we examined the possible cell cycle dependence of butyrate-induced cell death, its requirement for caspases, and its sensitivity to the anti-apoptotic Bcl-2 protein. Furthermore, we investigated a possible causal role of c-myc down-regulation in butyrate-induced cell death.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines and cell culture
CEM-C7H2 (29) is a glucocorticoid-sensitive subline of CCRF-CEM-C7 (28) . The generation of C7H2 sublines stably transfected with constructs allowing constitutive expression of the cowpox virus caspase inhibitor cytokine response modifier A (crmA; cell lines C7H2crmA-2E8, 2G10, 2H10; ref 30 ) has been described. C7H2–2A10 (31) and C7H2–2C8 (32) are subclones of the CEM-C7H2 cell line stably transfected with the tetracycline-controlled transcriptional transactivator, tTA (33) , or the reverse tTA, rtTA (34) , respectively. Cell lines C7H2tetBcl2-10E1, 9C3, and 9F3 (31) are stably transfected derivatives of the C7H2–2A10 cell line that express human bcl-2 under the control of tTA, i.e., expression is repressed in the presence of tetracycline (`tet-off' system). Cell lines C7H2tetMyc-B52 and D64 (32) and lines C7H2tetp16-1E10, 6E2, and 1D2 (35) are stably transfected derivatives of the C7H2–2C8 cell line that express human c-myc and p16/INK4A, respectively. In both instances, expression is controlled by rtTA, i.e., it is induced by tetracycline (`tet-on' system). All cell lines were grown in 5% CO2, saturated humidity, at 37°C in RPMI 1640 supplemented with 5–10% fetal or bovine calf serum (HyClone, Logan, Utah), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. For induction of c-myc or p16/INK4A (tet-on system), 200–300 ng/ml doxycycline was added to the media; for repression of transgenic bcl-2 (tet-off system), the cells were continuously exposed to 100 ng/ml doxycycline.

Inhibitory peptides and other reagents
Benzyloxycarbonyl-Val-Ala-Asp·fluoromethylketone (zVAD), benzyloxycarbonyl-Asp-Glu-Val-Asp·fluoromethylketone (DEVD), and benzyloxycarbonyl-Val-Glu-Ile-Asp·fluoromethylketone (VEID) were obtained from Enzyme System Products (Dublin, Calif.) and kept as a 10 mM stock solution in DMSO at -20°C. Sodium butyrate was prepared by titrating butyric acid (Fluka Chemie AG, Buchs, Switzerland) with sodium hydroxide to pH 7.3; trichostatin A, purified from Streptomyces hygroscopicus Y-50 (17) , was kindly provided by Dr. M. Yoshida. All other reagents, including sodium acetate, sodium propionate, sodium valerate, and propidium iodide, were from Sigma (Vienna, Austria) unless indicated otherwise.

Preparation of histones and analysis of histone acetylation
Histones were prepared from 107 or 108 cells. Acidic extraction of core histones was performed from whole cells with 0.3 M HCl after extraction of linker histones and HMG-proteins with 5% perchloric acid. For the extraction procedures with 5% perchloric acid as well as 0.3 M HCl, the pellet was taken up in 20 ml for a first extraction step and the extraction was repeated with 10 ml of the respective acids. In each extraction step, the pellets were subjected to homogenization in a Dounce homogenizer by 10 up-and-down strokes. After incubation for 30 min on ice, the homogenate was centrifuged for 15 min at 20,000 x g, 4°C. The supernatants of the two extractions with 0.3 M HCl were united and the core histones precipitated with 25% trichloric acid (final concentration). After incubation on ice for at least 1 h, the precipitate of the core histones was collected by centrifugation at 20,000 x g, 20 min, 4°C. The pellet was washed with HCl-acetone and dried in vacuo. Electrophoretic separation of the acetylated forms of histone H4 was performed in acidic urea-Triton X-100 polyacrylamide gels (12% T, 2.6% C, 8M urea) (36) .

Determination of proliferation and apoptosis
Degree of proliferation, as measured by 3H-thymidine uptake, was determined as previously detailed (29) . As an alternative, the MTT Cell Proliferation Kit I (Boehringer Mannheim, Vienna, Austria) was used according to the manufacturer's instructions. This colorimetric assay detects the cleavage of the tetrazolium salt MTT to a formazan dye that occurs only in metabolically active cells.

For detection and/or quantification of apoptosis, reduction of nuclear propidium iodide fluorescence together with forward/sideward light scattering analysis (37) , the annexin-V method (38) , and agarose gel analysis of DNA fragmentation (`DNA ladder'; ref 39 ) was used. FACS analysis of nuclear propidium iodide fluorescence and forward/sideward light scattering analysis have been described previously (40) . Briefly, 5 x 105 cells were permeabilized and stained with 750 µl propidium iodide (50 µg/ml in 0.1% Triton X-100/0.1% sodium citrate) and subjected to apoptosis analysis in an argon laser-equipped FACScan (Becton Dickinson, San Jose, Calif.), using either propidium iodide fluorescence intensity or forward/sideward light scattering as parameters. Cell debris and small particles were excluded from further analyses. Based on propidium iodide staining, cells in the sub-G1 marker window were considered to be apoptotic (see marker M1 in Fig. 1 A). Using forward/sideward light scattering as a parameter, apoptotic cells appear smaller (lower forward scatter values) and more granulated (higher sideward scatter values) than living cells (see markers R1 and R2 in Fig. 1A ). Annexin-V binding (38) was determined using the TACS annexin-V–FITC kit (TREVIGEN, Gaithersburg, Md.), as described by the manufacturer. Briefly, ~2.5 x 105 cells were incubated with FITC-labeled annexin-V and propidium iodide, washed, and analyzed on a FACScan as above (forward/sideward scatter, red and green fluorescence). As exemplified in Fig. 1A , living cells do not stain with annexin-V, are impermeable to propidium iodide, and hence appear in the lower left corner. Early apoptotic cells stain with annexin-V but are still impermeable to propidium iodide (lower right quadrant). Late apoptotic cells (and necrotic cells) are permeable for propidium iodide and hence locate to the upper right quadrant. For agarose gel detection of DNA fragmentation, DNA was prepared from 3 x 106 cells using the Genomic DNA Purification Kit (Promega, Madison, Wis.) as described by the manufacturer, separated on a 1.8% agarose gel, and stained with ethidium bromide.



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  		Figure 1. A) Butyrate induces apoptosis in CCRF-CEM leukemia cells. Data from four different apoptosis detection assays performed with untreated CEM-C7H2 cells and cells treated with 2 mM butyrate for 24 h are shown. Top two panels show forward/sideward light scattering (F/S) where apoptotic cells appear in marker R2. In the middle panels the simultaneously measured propidium iodide nuclear fluorescence intensity of Triton X-100 permeabilized cells (PI) is depicted; apoptotic cells are detected in the `sub-G1 peak' (marker M1). The two panels at the bottom correspond to intact cells stained with FITC-labeled annexin-V (Ax-V) and propidium iodide (in the absence of Triton X-100). Viable cells appear in the lower left, early apoptotic cells in the lower right, and late apoptotic (or necrotic) cells in the upper right, quadrants. The photograph at the bottom of panel A shows DNA extracted from butyrate-treated (But, second lane) or untreated (Co, third lane) CEM-C7H2 cells fractionated on an agarose gel and stained with ethidium bromide. In lanes 1 and 4, a DNA size marker was loaded for comparison. B) Time and dose dependence of butyrate-induced apoptosis. CEM-C7H2 cells were treated with butyrate at the concentrations and times indicated, and subjected to apoptosis determination by nuclear propidium iodide staining (top graph) or by the annexin-V method (bottom graph). Data from two experiments (mean ± standard deviations) are shown.

Cell cycle analysis
For cell cycle analyses, the propidium iodide method of Nicoletti et al. (37) was used as described above for determination of apoptosis by nuclear propidium iodide staining except that fluorescence intensity was plotted on a linear rather than a logarithmic scale.

Northern blot analysis
Northern analyses were performed as described previously (41) . Briefly, total RNA was extracted by a single-step extraction procedure, separated on formaldehyde-agarose gels, and blotted onto nitrocellulose membranes. The filters were hybridized with heat-denatured 32P-labeled human c-myc (kindly provided by Dr. M. Eilers; 42 ) or chicken GAPDH (donated by Dr. B. Auer; 43 ) cDNA probes, respectively, washed, and exposed to X-ray films with amplifying screens. Between hybridizations, blots were stripped with 0.1% sodium dodecyl sulfate at 80°C.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Butyrate and other histone deacetylase inhibitors induce apoptosis in human CCRF-CEM leukemia cells
To investigate possible anti-leukemic effects of sodium butyrate, we exposed CEM-C7H2 human leukemia cells to different amounts of butyrate and determined apoptosis levels after various incubation times using three different detection principles: reduction of nuclear propidium iodide fluorescence, forward/sideward light scattering, and annexin-V binding. As exemplified in Fig. 1A , all three methods, as well as a demonstration of nucleosomal DNA laddering as another hallmark of apoptosis, clearly showed that the cells underwent apoptotic death. In Fig. 1B , the dose and time dependence of sodium butyrate-induced apoptosis is demonstrated using two of the three FACS-based quantitative apoptosis assays (propidium iodide staining and annexin-V). Concentrations of up to 0.5 mM butyrate induced little if any apoptosis during the 48 h observation period. One to 4 mM butyrate increased apoptosis levels, first detectable after 12 h using the annexin-V method, followed by the propidium iodide technique, which showed significant cell death after 24 h. Whereas 1 mM butyrate caused only a low level of apoptosis, 2–4 mM killed essentially all cells after 36 to 48 h.

Since sodium butyrate is supposed to mediate many of its biological effects through inhibition of histone deacetylases, we determined histone acetylation in butyrate-treated CEM-C7H2 cells (Fig. 2 A). Treatment with 0.1 mM butyrate for 24 h (which has no effect on cell proliferation and survival; see Figs. 1 and 3 ) caused very little increase in histone H4 and H2B acetylation whereas 1 mM butyrate (which leads to cell death and reduced proliferation) entailed a marked increase in di-, tri-, and tetra-acetylated histones. To investigate whether other histone deacetylase inhibitors might also induce apoptosis in this system, we exposed CEM-C7H2 cells to three other short-chain fatty acids (acetate, valerate, and propionate) and to the structurally unrelated trichostatin A. As shown in Fig. 2B , these substances caused CEM-C7H2 apoptosis in concentrations that were closely related to those reported for their histone deacetylase inhibition (44) . Thus acetate, a poor inhibitor of deacetylases, induced very modest apoptosis and only at 32 mM. Valerate and propionate, better deacetylase inhibitors than acetate but weaker than butyrate, showed apoptosis induction starting at a four- to eightfold higher molarity (4 mM) than butyrate, whereas the potent deacetylase inhibitor trichostatin A was more than 1000-fold as cytotoxic as butyrate. The concerted findings suggested that apoptosis induction might have been the result of histone hyperacetylation.



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  		Figure 2. A) Butyrate induces histone hyperacetylation in CEM-C7H2 cells. Histone extracts prepared from untreated CEM-C7H2 (Co) and cells treated for 24 h with 0. 1 mM or 1. 0 mM butyrate were resolved on a 12% acidic urea-Triton X-100 polyacrylamide gel and stained with Coomassie blue R-250. B) Structurally distinct histone deacetylase inhibitors induce apoptosis in CEM-C7H2 human leukemia cells. CEM-C7H2 cells were treated with acetate, propionate, butyrate, or valerate for 24 h (top panel) or with trichostatin A for 24 and 48 h (bottom panel) at the concentrations indicated and subjected to apoptosis determination by nuclear propidium iodide staining and FACS analysis. The means ± standard deviations of two independent experiments (short-chain fatty acids) and a representative experiment in triplicate (trichostatin A) are shown.



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  		Figure 3. A) Butyrate causes accumulation of cells in the G2/M phase of the cell cycle. CEM-C7H2 cells treated with butyrate at the concentrations and times indicated were subjected to FACS cell cycle analyses as outlined in Material and Methods. Panels on the left show individual FACS plots, on the right a quantitative analysis of the same data in bar graphs (markers M1, 2, 3, and 4 in the plots correspond to apoptotic cells and cells in the G0/G1, S, and G2/M phases, respectively). Similar data were obtained in two repeat experiments. B) Butyrate causes reduction in cell proliferation as measured by 3H-thymidine uptake and MTT cleavage. CEM-C7H2 cells treated with butyrate at the concentrations and times indicated were subjected to 3H-thymidine uptake analyses (left panel) and the MTT cell proliferation assay (right panel). The means ± standard deviations from two experiments each performed in triplicate are shown.

Butyrate causes proliferation arrest with accumulation of cells in the G2/M phase of the cell cycle
Since chemotherapeutic drugs may act not only by inducing cell death but also by inhibiting proliferation, we studied the effect of butyrate on cell cycle progression and proliferation. FACS cell cycle analyses of butyrate-treated CEM-C7H2 cells revealed that the percentage of cells in G0/G1 markedly decreased in a time- and dose-dependent fashion, whereas cells in the G2/M phase of the cell cycle accumulated, followed by an increase in the percentage of apoptotic cells (Fig. 3 A). Concentrations of up to 0.5 mM butyrate showed little if any effect. One to 4 mM butyrate led to an increase in the number of cells in the G2/M phase and, at the same time, to an increased percentage of apoptotic cells and a decreased percentage of cells in the G0/G1 phase. This phenomenon became more pronounced with increasing butyrate concentrations and longer exposure times (from 16 to 24 h).

3H-Thymidine uptake (Fig. 3B , left panel) and MTT cleavage (Fig. 3B , right panel), two independent indicators for cell proliferation (Fig. 3B ), revealed a marked reduction largely coinciding with cell death. Thus, treatment for 12 h with any butyrate concentration tested or butyrate up to 0.5 mM for any time span tested neither induced cell death (Fig. 1B ) nor markedly reduced thymidine uptake or MTT cleavage (Fig. 3B ). Exposure to 1–4 mM butyrate for 24 h and longer led to marked apoptosis (Fig. 1B ), accumulation of cells in G2/M (Fig. 3A ), and inhibition of cell proliferation (Fig. 3B ). Thus, in these cells, induction of cell death, alterations of cell cycle distribution, and inhibition of proliferation occurred at similar butyrate concentrations and with similar kinetics.

Arrest in the G0/G1 phase of the cell cycle by expression of transgenic p16/INK4A inhibits butyrate-induced apoptosis
If butyrate-induced apoptosis occurred during or subsequent to accumulation of cells in the G2/M phase of the cell division cycle (as suggested by the data shown in Fig. 3A ), then forced G0/G1 cell cycle arrest might protect CEM-C7H2 cells from butyrate-induced cell death. To test this possibility, we treated three stably transfected CEM-C7H2 sublines (called C7H2tetp16-1E10, 6E2, and 1D2) that expressed exogenous p16/INK4A under tetracycline control with various concentrations of butyrate in the presence or absence of the tetracycline analog, doxycycline. In these experiments, doxycycline was added 24 h prior to butyrate to ascertain that the cells had already been arrested in G0/G1 when butyrate was added. As shown for the parental control (2C8) and one of the p16/INK4A-transfected clones (6E2) in the left panels of Fig. 4 A, induction of transgenic p16/INK4A by doxycycline caused accumulation of p16/INK4A-transfected 6E2 cells in the G0/G1 phase of the cell cycle, whereas untreated 6E2 and 2C8 control cells (with or without doxycycline) remained cycling. Treatment with 2 mM butyrate led to accumulation of cells in G2/M after 24 h and massive cell death after 48 h in all cells not expressing transgenic p16/INK4A. In contrast, butyrate-treated p16/INK4A-expressing cells (exemplified by 6E2 cells pretreated with doxycycline) failed to accumulate in G2/M and underwent only low levels of apoptosis. The two graphs on the right side of Fig. 4A summarize the effect of tetracycline-induced p16/INK4A expression on butyrate-mediated apoptosis in all three p16/INK4A-expressing cell lines treated with various concentrations of butyrate for 24 h and 48 h. Doxycycline (by inducing p16/INK4A-mediated G0/G1 arrest) markedly reduced the extent of apoptosis seen with 1 to 4 mM butyrate. In contrast, doxycycline had no effect on apoptosis in the untransfected CEM-C7H2 and transactivator-only transfected C7H2–2C8 parental control lines (data not shown). To determine whether the cells rescued by p16/INK4A were indeed viable and able to re-enter the cell cycle, the three p16/INK4A-transfected cell lines were treated with doxycycline for 15 h, washed to remove doxycycline (preliminary experiments have shown that cells treated this way arrest in G0/G1 for ~48–60 h), cultured for 9 h, treated with 2 mM butyrate for another 36 h, washed again to remove butyrate, cultured for another 60 h, and subjected to cell cycle analysis and apoptosis determination. As shown in Fig. 4B , the majority of the p16/INK4A-expressing cells had survived the 36 h butyrate exposure and re-entered the cell cycle, whereas the vast majority of cells not expressing p16/INK4A were killed. The combined data suggested that cells arrested in G0/G1 were resistant to the apoptosis-inducing butyrate effect.



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  		Figure 4. A) Expression of transgenic p16/INK4A prevents G2/M arrest and cell death in butyrate-treated CCRF-CEM leukemia cells. C7H2–2C8 (CEM-C7H2 derivatives stably transfected with rtTA) as control and C7H2tetp16-1E10, 6E2, and 1D2 (C7H2–2C8 derivatives stably transfected with a plasmid allowing tetracycline-induced p16/INK4A cDNA expression) were cultured in the presence or absence of 200 ng/ml doxycycline. After 24 h, the cultures were continued with or without the addition of butyrate in the concentrations indicated for another 24 to 48 h. Left panels show representative FACS cell cycle analyses of the 2C8 parental control cell line and p16/INK4A-transfected 6E2 cells cultured in the absence or presence of 2 mM butyrate with or without doxycycline pretreatment; 6E2 and 1D2 cells showed almost identical results. The graphs at the right summarize the percentages of apoptotic cells at various doses of butyrate, after 24 and 48 h, as determined by nuclear propidium iodide staining and FACS analyses. Since all three p16/INK4A-transfected cells gave essentially identical results, their data were pooled (referred to as C7H2tetp16). The means ± standard deviations from three independent experiments are shown, each with all three cell lines after 24 (top graph) and 48 h (bottom graph) butyrate exposure. In all three experiments, doxycycline showed no significant effect on the C7H2–2C8 parental control cell line (data not shown). B) CEM-C7H2 derivatives rescued from butyrate-induced cell death by p16/INK4A are viable and re-enter the cell cycle. p16/INK4A-transfected CEM-C7H2 cells transiently arrested in G0/G1 by a doxycycline pulse (see text) were cultured in the presence or absence of 2 mM butyrate for 36 h, washed, and subjected to cell cycle analysis and apoptosis determination after another 60 h. The panels show the FACS cell cycle analyses; C) p16/INK4A does not prevent butyrate-induced histone hyperacetylation. Histone H4 acetylation was determined in histone extracts prepared from C7H2tetp16-1D2 cells cultured for 23 h with or without doxycycline (lanes 1 and 2) and cells incubated with or without doxycycline for 23 h, followed by addition of 2 mM butyrate for another 5 h (lanes 3 and 4) or 37 h (lanes 5 and 6).

To determine whether the protective p16/INK4A effect might have resulted from prevention of deacetylase inhibition by butyrate, we investigated histone acetylation in cells treated with butyrate and expressing or not expressing p16/INK4A. The cdk inhibitor had no detectable effect on the extent of butyrate-induced hyperacetylation (Fig. 4C ), suggesting that histone hyperacetylation might be necessary (as suggested by the data shown in Fig. 2 ), but not sufficient, to induce cell death and that butyrate-induced apoptosis might be cell cycle dependent.

Caspases in butyrate-induced apoptosis
Most apoptosis pathways are associated with and depend on activation of caspases (45) , although which particular caspase (46) is activated may depend on the cell type and/or apoptosis inducer. To determine whether caspases are involved in butyrate-induced apoptosis, we treated CEM-C7H2 cells with butyrate in the presence of the tripeptide zVAD, which supposedly inhibits all known caspases (47) . As shown in Fig. 5 A, zVAD almost completely protected CEM-C7H2 cells from butyrate-induced apoptosis after 24 h, suggesting that zVAD-sensitive enzymes, most likely caspases, participated in this death response. However, as with other death responses, protection was not permanent and by 48 h was already considerably less pronounced. zVAD did not significantly alter the inhibitory effects of butyrate on thymidine uptake and MTT cleavage, indicating that these butyrate effects appear as caspase-independent, upstream events (Fig. 5A , middle and bottom graphs).



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  		Figure 5. A) zVAD interferes with butyrate-induced apoptosis but does not affect butyrate-mediated inhibition of proliferation. CEM-C7H2 cells were treated with butyrate in the indicated concentrations in the absence or presence of 100 µM zVAD for 24 (left graphs) or 48 h (right graphs) and subjected to apoptosis determination by nuclear propidium iodide staining and FACS analysis (top graphs) and to analysis of 3H-thymidine uptake (middle graphs) and MTT cleavage (bottom graphs). In the apoptosis assays, the means ± standard deviations from three experiments are shown, the proliferation assays by thymidine uptake or MTT cleavage were performed in triplicate. B) The cowpox virus caspase inhibitor crmA does not interfere with butyrate-induced apoptosis or inhibition of proliferation. CEM-C7H2 cells and three CEM-C7H2-derived cell lines stably transfected with a crmA expression construct (termed 2E8, 2G10, 2H10) were treated with butyrate in the indicated concentrations for 24 (left graphs) or 48 h (right graphs), subjected to apoptosis determination by nuclear propidium iodide staining and FACS analysis (top graphs), and to analysis of 3H-thymidine uptake (bottom graphs). The data for the three transfected cell lines were generated individually; however, since they were very similar, they were pooled for clarity (referred to as C7H2-crmA). A representative experiment with the three transfected cell lines (thymidine uptake analyses in triplicate) and the mean of two independent experiments with the parental CEM-C7H2 line are shown.

To address the possible involvement of lymphokine activator caspases (i.e., caspases 1, 4, and 5) and inducer caspases (such as caspase 8 and 10), we used the cowpox virus serpin crmA (cytokine response modifier A), shown to inhibit most of the above-mentioned caspases as well as granzyme B, an aspartate-specific serine protease also involved in apoptosis (48 49 50) . We treated three CEM-C7H2 sublines stably transfected with a crmA cDNA expression construct (termed 2E8, 2G10, and 2H10; 30) as well as the parental C7H2 line with butyrate. Although the crmA-expressing lines were completely resistant to apoptosis induced by antibodies to the CD95/fas/Apo-1 membrane protein (as we have previously shown) (30) , they were as sensitive as untransfected CEM-C7H2 to butyrate-induced apoptosis (Fig. 5B , top graphs). Thus, crmA-sensitive caspases did not seem to participate in this apoptotic response. As expected, crmA expression did not influence the effects of butyrate on cell proliferation as measured by thymidine uptake (Fig. 5B , bottom graphs).

To study a possible contribution of effector caspases, CEM-C7H2 cells were exposed to butyrate in the presence of the tetrapeptides DEVD and VEID, known inhibitors of effector caspases such as caspase 3, caspase 7 (DEVD), and caspase 6 (VEID; ref 51 ). In short-term assays (Table 1 ), both DEVD and VEID partially inhibited butyrate-induced apoptosis (as measured by nuclear propidium iodide fluorescence), although not to the same extent as zVAD, suggesting involvement of the above effector caspases. This finding supported the recent suggestion that butyrate induces a protein facilitating activation of caspase 3 in colorectal cancer and Jurkat lymphoid leukemia cells (52) .


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  		Table 1. Effect of caspase inhibitors on butyrate-induced apoptosisa

Bcl-2 interferes with butyrate-induced apoptosis but not butyrate inhibition of proliferation.
To determine whether butyrate-induced apoptosis in lymphatic leukemia cells is sensitive to the action of Bcl-2, we used three stably transfected CEM-C7H2 cell lines (termed C7H2tetBcl2-10E1, 9F3, and 9C3; 31) that express bcl-2 under the control of a tetracycline-repressible promoter. In the absence of the tetracycline analog doxycycline, these cell lines have been shown to express high levels of bcl-2 and to become almost completely resistant to dexamethasone exposure for 48 h, which kills these cells in the presence of doxycycline (31) . Induction of exogenous bcl-2 by withdrawal of doxycycline led to reduced cell death in butyrate-treated CEM cells (Fig. 6 , top graphs), although this inhibition was incomplete. To investigate whether Bcl-2 acts up- or downstream of inhibition of proliferation, we also determined 3H-thymidine uptake in these cells. As in the case of the caspase inhibitor zVAD (Fig. 5A ), bcl-2 overexpression did not alter butyrate-induced reduction in thymidine uptake (Fig. 6 , bottom graphs), placing the antiproliferative butyrate effect upstream of the site of Bcl-2 action.



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  		Figure 6. Transgenic Bcl-2 reduces butyrate-induced apoptosis but has no effect on inhibition of proliferation by butyrate. C7H2–2A10 (CEM-C7H2 derivatives stably transfected with tTA) as control and C7H2tetBcl2-10E1, 9C3, and 9F3 (C7H2–2A10 derivatives stably transfected with a plasmid allowing tetracycline-repressible human bcl-2 cDNA expression) were treated with butyrate at the indicated concentrations for 24 (left graphs) or 48 h (right graphs) and subjected to apoptosis determination by FACS analysis after nuclear propidium iodide staining (top graphs) and to analysis of 3H-thymidine uptake (bottom graphs). The FACS data for the three transfected cell lines were generated individually and, being very similar, were pooled for clarity (referred to as C7H2-tetBcl2). The extent of apoptosis in the C7H2–2A10 control cells was essentially the same in the presence or absence of doxycycline; hence, the data were not included in the graphs. Thymidine uptake is shown for 9C3 cells (as measured in triplicate ± SD).

C-myc does not prevent butyrate-induced apoptosis
Previous studies have shown that butyrate treatment reduces c-myc mRNA levels (4 , 10) , and c-myc down-regulation has been causally implicated in several death pathways (53) . We also observed c-myc mRNA steady-state down-regulation in butyrate-treated CEM-C7H2 cells (Fig. 7 , top). To determine a possible functional role of c-myc down-regulation by butyrate in the death pathway, we treated two stably transfected CCRF-CEM derivatives expressing exogenous c-myc under tetracycline control (called C7H2tetmyc-B52 and D64; 32) along with the parental rtTA-expressing C7H2–2C8 control line with various concentrations of butyrate in the presence and absence of doxycycline. As shown in Fig. 7 , butyrate-induced apoptosis was not prevented by induction of exogenous c-myc. On the contrary, transgenic c-myc sensitized the cells for butyrate-induced apoptosis: they died faster and at a lower butyrate concentration (0.5 mM) than in the absence of doxycycline-induced c-myc.



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  		Figure 7. Butyrate down-regulates c-myc steady-state mRNA levels in CCRF-CEM leukemia cells, but transgenic c-myc increases butyrate-induced apoptosis. Top: C7H2 cells were incubated with or without 2 mM butyrate for the indicated times and their RNA was subjected to Northern blot analysis using 32P-labeled cDNA probes for c-myc and, after stripping the filter, for GAPDH. Graphs below the Northern blots: C7H2–2C8 (CEM-C7H2 derivatives stably transfected with rtTA) as control and C7H2tetmyc-B52 and D64 (C7H2–2C8 derivatives stably transfected with a plasmid allowing tetracycline-inducible human c-myc cDNA expression) were treated with butyrate in the indicated concentrations for 24 (upper graph) or 48 (lower graph) h and subjected to apoptosis determination by FACS analysis after nuclear propidium iodide staining. The data for the two transfected cell lines were generated individually and, being very similar, were pooled for clarity (referred to as C7H2-myc). The extent of apoptosis in the C7H2–2C8 control cells was essentially the same in the presence or absence of doxycycline; hence, the data were not included in the graphs. The means ± standard deviations of the data of two independent experiments are shown.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study investigated the effects of the prototypic histone deacetylase inhibitor sodium butyrate on the CCRF-CEM lymphoid leukemia model. Butyrate induced typical apoptotic cell death (as detected by four different techniques), inhibited cell proliferation, and accumulated cells in the G2/M phase of the cell cycle. All three phenomena occurred with similar dose dependence and kinetics. The cell cycle arrest in G2/M was unexpected, because in most cells investigated butyrate caused a block in the G0/G1 phase (reviewed in refs 1 , 2 ). Apparently, CCRF-CEM cells are deficient in genes or regulatory mechanisms mediating this butyrate effect. In other systems, butyrate-induced G0/G1 arrest has been ascribed to c-myc repression (4 , 10) and/or p21/WAF1 up-regulation (24 25 26) . In CCRF-CEM cells, possible defects in the former mechanism are unlikely in view of the Northern blot experiments showing c-myc down-regulation by butyrate (Fig. 7) . Defects in p21/WAF1 regulation are unlikely also because this gene was normally induced in CCRF-CEM cells by p53 (40) . An alternative explanation for the failure to arrest in G0/G1 might be the reported deletion of the cell cycle inhibitor p16/INK4A in these cells (54) , which would make p16/INK4A an important target gene for butyrate-induced cell cycle arrest in G0/G1. When p16/INK4A was expressed as a tetracycline-inducible transgene in CCRF-CEM derivatives, the cells arrested in G0/G1 and became more resistant to butyrate. This suggested that loss of this putative tumor suppressor contributed to the malignant phenotype, but at the same time made the cells more sensitive to induction of apoptosis by histone deacetylase inhibition. Protection from cell death was permanent because the cells remained viable and entered the cell cycle when butyrate and doxycycline (used to induce p16/INK4A) were removed from the cultures. p16/INK4A did not detectably prevent butyrate-induced histone hyperacetylation, suggesting that the cells have to be in a `responsive' state, in this case the G2/M phase of the cell cycle to succumb to hyperacetylation-mediated cell death.

The signaling pathway leading to butyrate-induced cell death is largely unknown. As far as the terminal effector phase is concerned, we observed that this, like most other apoptosis pathways, involves zVAD-sensitive proteases/caspases with participation of DEVD/VEID-sensitive effector caspases. Similar to glucocorticoid- (30) and ceramide- (55) induced apoptosis, but distinct from that triggered by CD95/fas/Apo1 cross-linking (30) , crmA had no inhibitory effect on apoptosis in this leukemia model, arguing against a critical role of the CD95/fas/Apo1 system. Although both ends of the butyrate-induced death pathway are now better understood (initiation most likely by histone deacetylase inhibition and termination by caspase activation), little is known about the butyrate-regulated genes within the cascade beyond the implication of some candidates. Thus, bcl-2 has been shown to be down-regulated in several systems of butyrate-induced apoptosis (6 , 27) . Since CEM-C7H2 cells did not express bcl-2 mRNA at levels detectable by Northern analysis (data not shown), a possible regulation of this transcript could not be directly investigated. However, overexpression of transgenic bcl-2 did show some protective effect against butyrate-induced apoptosis, documenting that this apoptosis pathway is one of those affected by the `Bcl-2 rheostat' (56) . This raises the possibility that butyrate influences expression of corresponding genes in our lymphatic leukemia model as it does in other systems (6 , 27) . Another possible candidate gene, c-myc, whose repression has been implicated in several apoptosis pathways (53) , was down-regulated by butyrate in CCRF-CEM cells, as in other systems (4 , 10) . However, tetracycline-induced transgenic c-myc not only failed to rescue these cells from apoptosis, but even rendered them more sensitive to butyrate. Hence, at least in this model, c-myc down-regulation was not responsible for butyrate-induced cell death. We have observed an analogous phenomenon in the case of glucocorticoid-induced apoptosis, where endogenous c-myc was also down-regulated, yet transgenic c-myc enhanced GC-induced apoptosis rather than preventing it (32) . The combined data support the concept that c-myc has inherent proapoptotic properties (57) .


   ACKNOWLEDGMENTS
 
The authors thank Dr. M. Yoshida for providing trichostatin A, Ines Jaklitsch and Susanne Lobenwein for technical assistance, and M. Kat Occhipinti and Rajam Csordas-Iyer for editing the manuscript. Supported by grants from the Austrian Science Fund (SFB-F204, P-11946-Med), the Austrian National Bank (ÖNB-6156), and the University of Innsbruck.


   FOOTNOTES
 
Received for publication October 16, 1998. Revised for publication July 13, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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