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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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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: caspase • c-myc • bcl-2 • p16/INK4A • CCRF-CEM
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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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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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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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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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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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 U. Reisinger, S. Schwaiger, I. Zeller, B. Messner, R. Stigler, D. Wiedemann, T. Mayr, C. Seger, T. Schachner, V. M. Dirsch, et al. Leoligin, the major lignan from Edelweiss, inhibits intimal hyperplasia of venous bypass grafts Cardiovasc Res, June 1, 2009; 82(3): 542 - 549. [Abstract] [Full Text] [PDF]
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A. Scuto, M. Kirschbaum, C. Kowolik, L. Kretzner, A. Juhasz, P. Atadja, V. Pullarkat, R. Bhatia, S. Forman, Y. Yen, et al. The novel histone deacetylase inhibitor, LBH589, induces expression of DNA damage response genes and apoptosis in Ph- acute lymphoblastic leukemia cells Blood, May 15, 2008; 111(10): 5093 - 5100. [Abstract] [Full Text] [PDF]
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 V. R. Fantin and V. M. Richon Mechanisms of Resistance to Histone Deacetylase Inhibitors and Their Therapeutic Implications Clin. Cancer Res., December 15, 2007; 13(24): 7237 - 7242. [Abstract] [Full Text] [PDF]
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C. A. Banuelos, J. P. Banath, S. H. MacPhail, J. Zhao, T. Reitsema, and P. L. Olive Radiosensitization by the Histone Deacetylase Inhibitor PCI-24781 Clin. Cancer Res., November 15, 2007; 13(22): 6816 - 6826. [Abstract] [Full Text] [PDF]
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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]
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 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]
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 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]
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 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]
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 K. A. Strait, C. T. Warnick, C. D. Ford, B. Dabbas, E. H. Hammond, and S. J. Ilstrup Histone deacetylase inhibitors induce G2-checkpoint arrest and apoptosis in cisplatinum-resistant ovarian cancer cells associated with overexpression of the Bcl-2-related protein Bad Mol. Cancer Ther., April 1, 2005; 4(4): 603 - 611. [Abstract] [Full Text] [PDF]
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 D. Bandyopadhyay, A. Mishra, and E. E. Medrano Overexpression of Histone Deacetylase 1 Confers Resistance to Sodium Butyrate-Mediated Apoptosis in Melanoma Cells through a p53-Mediated Pathway Cancer Res., November 1, 2004; 64(21): 7706 - 7710. [Abstract] [Full Text] [PDF]
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R. L. Piekarz, R. W. Robey, Z. Zhan, G. Kayastha, A. Sayah, A. H. Abdeldaim, S. Torrico, and S. E. Bates T-cell lymphoma as a model for the use of histone deacetylase inhibitors in cancer therapy: impact of depsipeptide on molecular markers, therapeutic targets, and mechanisms of resistance Blood, June 15, 2004; 103(12): 4636 - 4643. [Abstract] [Full Text] [PDF]
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 J.-J. Liu, J. Zhang, S. Ramanan, J. Julian, D. D. Carson, and S. C. Hooi Heparin/heparan sulfate interacting protein plays a role in apoptosis induced by anticancer drugs Carcinogenesis, June 1, 2004; 25(6): 873 - 879. [Abstract] [Full Text] [PDF]
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X. D. Zhang, S. K. Gillespie, J. M. Borrow, and P. Hersey The histone deacetylase inhibitor suberic bishydroxamate regulates the expression of multiple apoptotic mediators and induces mitochondria-dependent apoptosis of melanoma cells Mol. Cancer Ther., April 1, 2004; 3(4): 425 - 435. [Abstract] [Full Text] [PDF]
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L. Catley, E. Weisberg, Y.-T. Tai, P. Atadja, S. Remiszewski, T. Hideshima, N. Mitsiades, R. Shringarpure, R. LeBlanc, D. Chauhan, et al. NVP-LAQ824 is a potent novel histone deacetylase inhibitor with significant activity against multiple myeloma Blood, October 1, 2003; 102(7): 2615 - 2622. [Abstract] [Full Text] [PDF]
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M. Di Padova, T. Bruno, F. De Nicola, S. Iezzi, C. D'Angelo, R. Gallo, D. Nicosia, N. Corbi, A. Biroccio, A. Floridi, et al. Che-1 Arrests Human Colon Carcinoma Cell Proliferation by Displacing HDAC1 from the p21WAF1/CIP1 Promoter J. Biol. Chem., September 19, 2003; 278(38): 36496 - 36504. [Abstract] [Full Text] [PDF]
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M. J. Peart, K. M. Tainton, A. A. Ruefli, A. E. Dear, K. A. Sedelies, L. A. O'Reilly, N. J. Waterhouse, J. A. Trapani, and R. W. Johnstone Novel Mechanisms of Apoptosis Induced by Histone Deacetylase Inhibitors Cancer Res., August 1, 2003; 63(15): 4460 - 4471. [Abstract] [Full Text] [PDF]
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 J. L. Merchant, L. Bai, and M. Okada ZBP-89 Mediates Butyrate Regulation of Gene Expression J. Nutr., July 1, 2003; 133(7): 2456S - 2460. [Abstract] [Full Text] [PDF]
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C. Yu, M. Rahmani, J. Almenara, M. Subler, G. Krystal, D. Conrad, L. Varticovski, P. Dent, and S. Grant Histone Deacetylase Inhibitors Promote STI571-mediated Apoptosis in STI571-sensitive and -resistant Bcr/Abl+ Human Myeloid Leukemia Cells Cancer Res., May 1, 2003; 63(9): 2118 - 2126. [Abstract] [Full Text] [PDF]
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S. Skov, K. Rieneck, L. F. Bovin, K. Skak, S. Tomra, B. K. Michelsen, and N. Odum Histone deacetylase inhibitors: a new class of immunosuppressors targeting a novel signal pathway essential for CD154 expression Blood, February 15, 2003; 101(4): 1430 - 1438. [Abstract] [Full Text] [PDF]
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 W. J. M. Mackus, S. M. A. Lens, R. H. Medema, M. J. Kwakkenbos, L. M. Evers, M. H. J. v. Oers, R. A. W. v. Lier, and E. Eldering Prevention of B cell antigen receptor-induced apoptosis by ligation of CD40 occurs downstream of cell cycle regulation Int. Immunol., September 1, 2002; 14(9): 973 - 982. [Abstract] [Full Text] [PDF]
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M. V. Blagosklonny, R. Robey, D. L. Sackett, L. Du, F. Traganos, Z. Darzynkiewicz, T. Fojo, and S. E. Bates Histone Deacetylase Inhibitors All Induce p21 but Differentially Cause Tubulin Acetylation, Mitotic Arrest, and Cytotoxicity Mol. Cancer Ther., September 1, 2002; 1(11): 937 - 941. [Abstract] [Full Text] [PDF]
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S. Taniura, H. Kamitani, T. Watanabe, and T. E. Eling Transcriptional Regulation of Cyclooxygenase-1 by Histone Deacetylase Inhibitors in Normal Human Astrocyte Cells J. Biol. Chem., May 3, 2002; 277(19): 16823 - 16830. [Abstract] [Full Text] [PDF]
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A. J. Burgess, S. Pavey, R. Warrener, L.-J. K. Hunter, T. J. Piva, E. A. Musgrove, N. Saunders, P. G. Parsons, and B. G. Gabrielli Up-Regulation of p21WAF1/CIP1 by Histone Deacetylase Inhibitors Reduces Their Cytotoxicity Mol. Pharmacol., October 1, 2001; 60(4): 828 - 837. [Abstract] [Full Text] [PDF]
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 M. Inman, G.-C. Perng, G. Henderson, H. Ghiasi, A. B. Nesburn, S. L. Wechsler, and C. Jones Region of Herpes Simplex Virus Type 1 Latency-Associated Transcript Sufficient for Wild-Type Spontaneous Reactivation Promotes Cell Survival in Tissue Culture J. Virol., April 15, 2001; 75(8): 3636 - 3646. [Abstract] [Full Text] [PDF]
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 R. W. Johnstone, M. Gerber, T. Landewe, A. Tollefson, W. S. Wold, and A. Shilatifard Functional Analysis of the Leukemia Protein ELL: Evidence for a Role in the Regulation of Cell Growth and Survival Mol. Cell. Biol., March 1, 2001; 21(5): 1672 - 1681. [Abstract] [Full Text] [PDF]
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 M. TONKO, M. J. AUSSERLECHNER, D. BERNHARD, A. HELMBERG, and R. KOFLER Gene expression profiles of proliferating vs. G1/G0 arrested human leukemia cells suggest a mechanism for glucocorticoid-induced apoptosis FASEB J, March 1, 2001; 15(3): 693 - 699. [Abstract] [Full Text] [PDF]
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W.-G. Zhu, R. R. Lakshmanan, M. D. Beal, and G. A. Otterson DNA Methyltransferase Inhibition Enhances Apoptosis Induced by Histone Deacetylase Inhibitors Cancer Res., February 1, 2001; 61(4): 1327 - 1333. [Abstract] [Full Text]
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