(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
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ABSTRACT
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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
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INTRODUCTION
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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.
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MATERIALS AND METHODS
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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. C7H22A10 (31)
and C7H22C8
(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 C7H22A10 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
C7H22C8 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 510%
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), 200300
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-VFITC 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.
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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
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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,
24 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.
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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 14 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 C7H22C8 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 ~4860 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.
C7H22C8 (CEM-C7H2 derivatives stably transfected with rtTA) as
control and C7H2tetp16-1E10, 6E2, and 1D2 (C7H22C8
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
C7H22C8 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).
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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)
.
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. C7H22A10 (CEM-C7H2
derivatives stably transfected with tTA) as control and
C7H2tetBcl2-10E1, 9C3, and 9F3 (C7H22A10 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 C7H22A10 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 C7H22C8 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: C7H22C8 (CEM-C7H2 derivatives
stably transfected with rtTA) as control and C7H2tetmyc-B52
and D64 (C7H22C8 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 C7H22C8 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
|
|---|
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
|
|---|
-
Csordas, A. (1995) Toxicology of butyrate and short-chain fatty acids. Hill, M. J. eds. Role of Gut Bacteria in Human Toxicology and Pharmacology ,105-127 Taylor and Francis London.
-
Kruh, J., Defer, N., Tichonicky, L. (1995) Effects of butyrate on cell proliferation and gene expression. Cummings, J. H. Rombeau, J. L. Sakata, T. eds. Physiology and Clinical Aspects of Short-chain Fatty Acids ,275-288 Cambridge University Press London.
-
Hague, A., Elder, D. J., Hicks, D. J., Paraskeva, C. (1995) Apoptosis in colorectal tumor cells: induction by the short-chain fatty acids butyrate, propionate and acetate and by bile salt deoxycholate. Int. J. Cancer 60,400-406[Medline]
-
Heruth, D. P., Zirnstein, G. W., Bradley, J. F., Rothberg, P. G. (1993) Sodium butyrate causes an increase in the block to transcriptional elongation in the c-myc gene in SW837 rectal carcinoma cells. J. Biol. Chem. 268,20466-20472[Abstract/Free Full Text]
-
Soldatenkov, V. A., Prasad, S., Voloshin, Y., Dritschilo, A. (1998) Sodium butyrate induces apoptosis and accumulation of ubiquitinated proteins in human breast carcinoma cells. Cell Death Differ 5,307-312[Medline]
-
Mandal, M., Kumar, R. (1996) Bcl-2 expression regulates sodium butyrate-induced apoptosis in human MCF-7 breast cancer cells. Cell Growth & Differ 7,311-318[Abstract]
-
Wang, X. M., Wang, X., Li, J., Evers, B. M. (1998) Effects of 5-azacitidine and butyrate on differentiation and apoptosis of hepatic cancer cell lines. Ann. Surg. 227,922-931[Medline]
-
Urbano, A., Koc, Y., Foss, F. M. (1998) Arginine butyrate downregulates p210 bcr-abl expression and induces apoptosis in chronic myelogenous leukemia cells. Leukemia 12,930-936[Medline]
-
Garcia-Bermejo, L., Vilaboa, N. E., Perez, C., Galan, A., De Blas, E., Aller, P. (1997) Modulation of heat-shock protein 70 (HSP70) gene expression by sodium butyrate in U-937 promonocytic cells: relationships with differentiation and apoptosis. Exp. Cell Res. 236,268-274[Medline]
-
Buckley, A. R., Leff, M. A., Buckley, D. J., Magnuson, N. S., De Jong, G., Gout, P. W. (1996) Alterations in pim-1 and c-myc expression associated with butyrate-induced growth factor dependence in autonomous rat NB2 lymphoma cells. Cell Growth & Differ 7,1713-1721[Abstract]
-
Conley, B. A., Egorin, M. J., Tait, N., Rosen, D. M., Sausville, E. A., Dover, G., Fram, R. J., Van Echo, D. A. (1998) Phase I study of the orally administered butyrate prodrug, tributyrin, in patients with solid tumors. Clin. Cancer Res. 4,629-634[Abstract]
-
Novogrodsky, A., Dvir, A., Ravid, A., Shkolnik, T., Stenzel, K. H., Rubin, A. L. Z. R. (1983) Effect of polar organic compounds on leukemic cells. Butyrate-induced partial remission of acute myelogenous leukemia in a child. Cancer 51,9-14[Medline]
-
Miller, A. A., Kurschel, E., Osieka, R., Schmidt, C. G. (1987) Clinical pharmacology of sodium butyrate in patients with acute leukemia. Eur. J. Clin. Oncol. 23,1283-1287
-
Kasukabe, T., Rephaeli, A., Honma, Y. (1997) An anti cancer derivative of butyric acid (pivalyloxymethyl butyrate) and daunorubicin cooperatively prolong survival of mice inoculated with monocytic leukaemia cells. Br. J. Cancer 75,850-854[Medline]
-
Riggs, M. G., Whittaker, R. G., Neumann, J. R., Ingram, V. M. (1977) n-Butyrate causes histone modification in HeLa and Friend erythroleukemia cells. Nature (London) 268,462-464[Medline]
-
Boffa, L. C., Vidali, G., Mann, R. S., Allfrey, V. G. (1978) Suppression of histone deacetylation in vivo and in vitro by sodium butyrate. J. Biol. Chem. 253,3364-3366[Abstract/Free Full Text]
-
Yoshida, M., Kijima, M., Akita, M., Beppu, T. (1990) Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J. Biol. Chem. 265,17174-17179[Abstract/Free Full Text]
-
Wolffe, A. P., Pruss, D. (1996) Targeting chromatin disruption: transcription regulators that acetylate histones. Cell 84,817-819[Medline]
-
Grunstein, M. (1997) Histone acetylation in chromatin structure and transcription. Nature (London) 389,349-352[Medline]
-
Pazin, M. J., Kadonaga, J. T. (1997) What's up and down with histone deacetylation and transcription. Cell 89,325-328[Medline]
-
Struhl, K. (1998) Histone acetylation and transcriptional regulatory mechanisms. Genes Dev 12,599-606[Free Full Text]
-
Perrine, S. P., Ginder, G. D., Faller, D. V., Dover, G. H., Ikuta, T., Witkowska, H. E., Cai, S.-P., Vichinsky, E. P., Olivieri, N. F. (1993) A short-term trial of butyrate to stimulate fetal-globin gene expression in the ß-globin disorders. N. Engl. J. Med. 328,81-86[Abstract/Free Full Text]
-
McCaffrey, P. G., Newsome, D. A., Fibach, E., Yoshida, M., Su, M. S. S. (1997) Induction of gamma-globin by histone deacetylase inhibitors. Blood 90,2075-2083[Abstract/Free Full Text]
-
Archer, S. Y., Meng, S. F., Shei, A., Hodin, R. A. (1998) p21WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells. Proc. Natl. Acad. Sci. USA 95,6791-6796[Abstract/Free Full Text]
-
Nakano, K., Mizuno, T., Sowa, Y., Orita, T., Yoshino, T., Okuyama, Y., Fujita, T., Ohtani-Fujita, N., Matsukawa, Y., Tokino, T., Yamagishi, H., Oka, T., Nomura, H., Sakai, T. (1997) Butyrate activates the WAF1/Cip1 gene promoter through Sp1 sites in a p53-negative human colon cancer cell line. J. Biol. Chem. 272,22199-22206[Abstract/Free Full Text]
-
Janson, W., Brandner, G., Siegel, J. (1997) Butyrate modulates DNA-damage-induced p53 response by induction of p53-independent differentiation and apoptosis. Oncogene 15,1395-1406[Medline]
-
Hague, A., Díaz, G. D., Hicks, D. J., Krajewski, S., Reed, J. C., Paraskeva, C. (1997) Bcl-2 and bak may play a pivotal role in sodium butyrate-induced apoptosis in colonic epithelial cells; however, overexpression of bcl-2 does not protect against bak-mediated apoptosis. Int. J. Cancer 72,898-905[Medline]
-
Norman, M. R., Thompson, E. B. (1977) Characterization of a glucocorticoid-sensitive human lymphoid cell line. Cancer Res 37,3785-3791[Abstract/Free Full Text]
-
Strasser-Wozak, E. M. C., Hattmannstorfer, R., Hála, M., Hartmann, B. L., Fiegl, M., Geley, S., Kofler, R. (1995) Splice site mutation in the glucocorticoid receptor gene causes resistance to glucocorticoid-induced apoptosis in a human acute leukemic cell line. Cancer Res 55,348-353[Abstract/Free Full Text]
-
Geley, S., Hartmann, B. L., Kapelari, K., Egle, A., Villunger, A., Heidacher, D., Greil, R., Auer, B., Kofler, R. (1997) The interleukin 1ß-converting enzyme inhibitor crmA prevents Apo1/fas- but not glucocorticoid-induced poly(ADP-ribose) polymerase cleavage and apoptosis in lymphoblastic leukemia cells. FEBS Lett 402,36-40[Medline]
-
Hartmann, B. L., Geley, S., Löffler, M., Hattmannstorfer, R., Strasser-Wozak, E. M. C., Auer, B., Kofler, R. (1999) Bcl-2 interferes with the execution phase, but not upstream events, in glucocorticoid-induced leukemia apoptosis. Oncogene 18,713-719[Medline]
-
Löffler, M., Tonko, M., Hartmann, B. L., Bernhard, D., Geley, S., Helmberg, A., Kofler, R. (1999) c-myc does not prevent glucocorticoid-induced apoptosis of human leukemic lymphoblasts. Oncogene 18,4626-4631[Medline]
-
Gossen, M., Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89,5547-5551[Abstract/Free Full Text]
-
Gossen, M., Freundlieb, S., Bender, G., Müller, G., Hillen, W., Bujard, H. (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268,1766-1769[Abstract/Free Full Text]
-
Ausserlechner, M. J., Obexer, P., Hartmann, B. L.,
Geley, S., and Kofler, R. (1999) Tetracycline-regulated p16/INK4A
induces G1/Go arrest and differentiation in human p16/INK4A-deficient
malignant T lymphoblasts. In preparation
-
Zweidler, A. (1978) Resolution of histones by polyacrylamide
gel electrophoresis in presence of nonionic detergents. Methods Cell Biol. 17,223-233[Medline]
-
Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F., Riccardi, C. (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139,271-279[Medline]
-
Martin, S. J., Reutelingsperger, C. P. M., Green, D. R. (1996) Annexin-V: a specific probe for apoptotic cells. Cotter, T. G. Martin, S. J. eds. Techniques in Apoptosis ,107-120 Portland Press Ltd. London.
-
Gong, J., Traganos, F., Darzynkiewicz, Z. (1994) A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal. Biochem. 218,314-319[Medline]
-
Geley, S., Hartmann, B. L., Hattmannstorfer, R., Löffler, M., Ausserlechner, M. J., Bernhard, D., Sgonc, R., Strasser-Wozak, E. M. C., Ebner, M., Auer, B., Kofler, R. (1997) P53-induced apoptosis in the human T-ALL cell line CCRF-CEM. Oncogene 15,2429-2437[Medline]
-
Geley, S., Hartmann, B. L., Hala, M., Strasser-Wozak, E. M. C., Kapelari, K., Kofler, R. (1996) Resistance to glucocorticoid-induced apoptosis in human T-cell acute lymphoblastic leukemia CEM-C1 cells is due to insufficient glucocorticoid receptor expression. Cancer Res 56,5033-5038[Abstract/Free Full Text]
-
Eilers, M., Picard, D., Yamamoto, K. R., Bishop, J. M. (1989) Chimaeras of myc oncoprotein and steroid receptors cause hormone-dependent transformation of cells. Nature (London) 340,66-68[Medline]
-
Dugaiczyk, A., Haron, J. A., Stone, E. M., Dennison, O. E., Rothblum, K. N., Schwartz, R. J. (1983) Cloning and sequencing of a deoxyribonucleic acid copy of glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acid isolated from chicken muscle. Biochemistry 22,1605-1613[Medline]
-
McBain, J. A., Eastman, A., Nobel, C. S., Mueller, G. C. (1997) Apoptotic death in adenocarcinoma cell lines induced by butyrate and other histone deacetylase inhibitors. Biochem. Pharmacol. 53,1357-1368[Medline]
-
Salvesen, G. S., Dixit, V. M. (1997) Caspases: intracellular signaling by proteolysis. Cell 91,443-446[Medline]
-
Alnemri, E. S., Livingston, D. J., Nicholson, D. W., Salvesen, G., Thornberry, N. A., Wong, W. W., Yuan, J. Y. (1996) Human ICE/CED-3 protease nomenclature. Cell 87,171-171[Medline]
-
Villa, P., Kaufmann, S. H., Earnshaw, W. C. (1997) Caspases and caspase inhibitors. Trends Biochem. Sci. 22,388-393[Medline]
-
Quan, L. T., Caputo, A., Bleackley, R. C., Pickup, D. J., Salvesen, G. S. (1995) Granzyme B is inhibited by the cowpox virus serpin cytokine response modifier A. J. Biol. Chem. 270,10377-10379[Abstract/Free Full Text]
-
Zhou, Q., Snipas, S., Orth, K., Muzio, M., Dixit, V. M., Salvesen, G. S. (1997) Target protease specificity of the viral serpin CrmAanalysis of five caspases. J. Biol. Chem. 272,7797-7800[Abstract/Free Full Text]
-
Kamada, S., Funahashi, Y., Tsujimoto, Y. (1997) Caspase-4 and caspase-5, members of the ICE/CED-3 family of cysteine proteases, are CrmA-inhibitable proteases. Cell Death Differ 4,473-478
-
Thornberry, N. A., Ranon, T. A., Pieterson, E. P., Rasper, D. M., Timkey, T., Garcia-Calvo, M., Houtzager, V. M., Nordstrom, P. A., Roy, S., Vaillancourt, J. P., Chapman, K. T., Nicholson, D. W. (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme Bfunctional relationships established for key mediators of apoptosis. J. Biol. Chem. 272,17907-17911[Abstract/Free Full Text]
-
Medina, V., Edmonds, B., Young, G. P., James, R., Appleton, S., Zalewski, P. D. (1997) Induction of caspase-3 protease activity and apoptosis by butyrate and trichostatin A (inhibitors of histone deacetylase): dependence on protein synthesis and synergy with a mitochondrial cytochrome c-dependent pathway. Cancer Res 57,3697-3707[Abstract/Free Full Text]
-
Thompson, E. B. (1998) The many roles of c-Myc in apoptosis. Annu. Rev. Physiol. 60,575-600[Medline]
-
Otsuki, T., Clark, H. M., Wellmann, A., Jaffe, E. S., Raffeld, M. (1995) Involvement of CDKN2 (p16INK4A/MTS1) and p15INK4B/MTS2 in human leukemias and lymphomas. Cancer Res 55,1436-1440[Abstract/Free Full Text]
-
Geley, S., Hartmann, B. L., Kofler, R. (1997) Ceramides induce a form of apoptosis in human acute lymphoblastic leukemia cells that is inhibited by Bcl-2, but not by CrmA. FEBS Lett 400,15-18[Medline]
-
Oltvai, Z. N., Korsmeyer, S. J. (1994) Checkpoints of dueling dimers foil death wishes. Cell 79,189-192[Medline]
-
Evan, G., Littlewood, T. (1998) A matter of life and cell death. Science 281,1317-1322[Abstract/Free Full Text]
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S. Liu, R. B. Klisovic, T. Vukosavljevic, J. Yu, P. Paschka, L. Huynh, J. Pang, P. Neviani, Z. Liu, W. Blum, et al.
Targeting AML1/ETO-Histone Deacetylase Repressor Complex: A Novel Mechanism for Valproic Acid-Mediated Gene Expression and Cellular Differentiation in AML1/ETO-Positive Acute Myeloid Leukemia Cells
J. Pharmacol. Exp. Ther.,
June 1, 2007;
321(3):
953 - 960.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
D. Bernhard, A. Rossmann, B. Henderson, M. Kind, A. Seubert, and G. Wick
Increased Serum Cadmium and Strontium Levels in Young Smokers: Effects on Arterial Endothelial Cell Gene Transcription
Arterioscler. Thromb. Vasc. Biol.,
April 1, 2006;
26(4):
833 - 838.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
M. R. Acharya, A. Sparreboom, J. Venitz, and W. D. Figg
Rational Development of Histone Deacetylase Inhibitors as Anticancer Agents: A Review
Mol. Pharmacol.,
October 1, 2005;
68(4):
917 - 932.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
X.-J. Han, J.-K. Chae, M.-J. Lee, K.-R. You, B.-H. Lee, and D.-G. Kim
Involvement of GADD153 and Cardiac Ankyrin Repeat Protein in Hypoxia-induced Apoptosis of H9c2 Cells
J. Biol. Chem.,
June 17, 2005;
280(24):
23122 - 23129.
[Abstract]
[Full Text]
[PDF]
|
 |
|