From the Dipartimento di Scienze e Tecnologie
Biomediche, Sezione di Biologia, Universita' di Udine, P.le Kolbe 4, 33100 Udine Italy, ** Laboratorio Nazionale Consorzio
Interuniversitario Biotecnologie AREA Science Park, Padriciano 99 34142 Trieste Italy,
MMNP-Unit Experimental Oncology CRO-IRCCS
National Cancer Institute Via Pedemontana Occ. 12 33081 Aviano (PN)
Italy, and § MATI Center of Excellence, Universita' di
Udine. P.le Kolbe 4, 33100 Udine Italy
Received for publication, December 23, 2002, and in revised form, January 29, 2003
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ABSTRACT |
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Histone deacetylase activity is potently
inhibited by hydroaximc acid derivatives such as suberoylanilide
hydroxamic acid (SAHA) and trichostatin-A (TSA). These inhibitors
specifically induce differentiation/apoptosis of transformed cells
in vitro and suppress tumor growth in vivo.
Because of its low toxicity, SAHA is currently evaluated in clinical
trials for the treatment of cancer. SAHA and TSA induce apoptosis,
which is characterized by mitochondrial stress, but so far, the
critical elements of this apoptotic program remain poorly defined. To
characterize in more detail this apoptotic program, we used human cell
lines containing alterations in important elements of apoptotic
response such as: p53, Bcl-2, caspase-9, and caspase-3. We demonstrate that caspase-9 is critical for apoptosis induced by SAHA and TSA and
that efficient proteolytic activation of caspase-2, caspase-8, and caspase-7 strictly depends on caspase-9. Bcl-2 efficiently antagonizes cytochrome c release and apoptosis in response
to both histone deacetylase inhibitors. We provide evidences that translocation into the mitochondria of the Bcl-2 family member Bid
depends on caspase-9 and that this translocation is a late event during
TSA-induced apoptosis. We also demonstrate that the susceptibility to TSA- and SAHA-induced cell death is regulated by p53.
Histone acetyl transferases and histone deacetylases
(HDACs)1 are emerging as important components
that affect the dynamics of chromatin
folding during gene transcription (1). HDACs catalyze the hydrolyisis
of acetyl groups from amino-terminal lysine residues of the nucleosomal
core histones (1, 2). Histone deacetylases inhibitors (HDIs) are
promising agents for anticancer therapy; they exhibit strong antitumor
activities in vivo with low toxicity in preclinical studies.
HADC inhibitors belong to a heterogenous class of compounds that
includes derivatives of short chain fatty acids, hydroxamic acids,
cyclic tetrapetides, and benzamides. Among the hydroxamic acids,
trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) are
commonly used inhibitors of HDACs (3, 4).
Numerous anti-proliferative effects have been reported for TSA and
SAHA, including induction of G0/G2 cell cycle
arrest, differentiation, and selective apoptosis of transformed cells
(5-8). SAHA, in particular, shows strong anti-proliferative effects
but low toxicity in vivo and is currently under clinical
trials for the treatment of solid and hematological tumors (3, 4).
The cystein proteases, which belong to the family of caspases, play a
critical role in the apoptotic response (9-11). Several anticancer
drugs exert their antineoplastic activity by inducing tumor cell
apoptosis. Various of these antitumor drugs trigger mitochondrial
stress that can lead to apoptosome-mediated caspase-9 activation.
Caspase-9 activates the effectors caspase-3 and -7, which then trigger
cell fragmentation by cleaving selected death substrates and also
process different caspases, thus leading to the generation of the
amplification loop (12, 13).
An alternative apoptotic pathway is triggered by the cell surface death
receptor (extrinsic pathway), which includes caspase-8 and caspase-10
as apical caspases (10, 11, 14). However by cleaving Bid, a Bcl-2
family member, these caspases can induce mitochondria permeabilization
and activation of the amplifier function of the apoptosome (15).
Chemoresistance is frequently caused by aberrant apoptosis that
in some instances has been related to defects in caspase activation (16, 17). Therefore, the definition of the apoptotic pathway that is
induced by a particular anticancer drug is important to design
therapeutic trials and clinical treatment.
The mechanism of HDI-induced apoptosis has only been marginally
addressed. The role of the tumor suppressor p53 in the HDI-triggered apoptosis is not clear. Some reports have suggested an effect of p53 in
this apoptotic response (18-21), whereas other studies have pointed to
a p53-independent apoptotic response (22-24). Mitochondrial stress and release of cytochrome c mark the apoptotic
response to HDIs (5, 23, 24, 26), and changes in the expression of
Bcl-2 family members such as Bcl-2, Bax, or Bad have been detected in
some cell lines (26, 27). Caspase-3 activation was reported by
different studies (5, 23, 24, 26), but cell death after SAHA treatment
was observed also in the presence of pancaspase inhibitor
zVAD-fmk (23). However, the same study suggested that a caspase not
efficiently inhibited by zVAD-fmk could be involved in transducing
the apoptotic signal triggered by SAHA (23).
Our work was designed to identify the critical elements involved in
transducing HDI-induced apoptotic signals. Human cell lines containing
known mutations in key elements of the apoptotic pathway allowed us to
elucidate caspase-9 requirements in the apoptotic response to HDIs. We
show that HDI-induced proteolytic activation of caspase-2, caspase-8,
and caspase-7 was strictly dependent on caspase-9. Moreover, HDIs
provoke, late during apoptosis, translocation of the Bcl-2 family
member Bid into the mitochondria, and this translocation also depends
on caspase-9. We also show that Bcl-2 efficiently antagonizes
cytochrome c release and apoptosis in response to TSA.
Finally, we demonstrate that the susceptibility to cell death in
response to TSA and SAHA treatment is regulated by p53.
Culture Conditions and Drug Treatment--
IMR90, IMR90-E1A,
IMR90-E1A/C9DN (31) MCF-7/caspase-3 wt (C3WT), MCF-7/caspase-3
CI (C3CI), MCF-7/NEO, and MCF-7/C9DN were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum, penicillin
(100 units/ml), glutamine (2 mM), and streptomycin (100 µg/ml) at 37 °C in 5% CO2 atmosphere. Cells were
cultured for 12-60 h with 1 µM TSA, for 24-48 h with 2.5 µM SAHA, for 44 h with 40 µM
colchicine, for 44 h with 14 µM
1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine
or for 44 h with 10 µM daunorubicin. Early
passage BJ normal human foreskin fibroblasts were maintained in minimum
essential medium with Earle's salts supplemented with non-essential
amino acids and 10% fetal bovine serum (Invitrogen) at 37 °C in the
presence of 5% CO2.
In all trypan blue exclusion assays, 480-2400 cells, from three
independent samples, were counted for each data point; data were
calculated as the means of at least three independent experiments. The
number of apoptotic cells (blue cells) was expressed as a percentage of
the total cell number.
Transfection, Microinjection, and Time Lapse Analysis--
For
Bcl-2 survival activity, transfections were performed using the calcium
phosphate precipitation method. Cells were seeded at 1.2 × 104 cell/ml, and after 18 h, they were co-transfected
with 2 µg of Bcl-2 expression plasmid or NEO expression plasmid were
together with 200 ng of pEGFP-N1 (Invitrogen) to score the transfected cells in vivo. After 24 h, transfected cells
were incubated with 1 µM TSA.
Microinjection was performed using the automated injection
system (Zeiss, Germany) as described previously (28). Cells were injected with 10 ng of pEGFPN1Bid for 0.5 s at a
constant pressure of 50 hPA. Time-lapse studies were performed using a
laser scan microscopy Leica TCS-SP in a 5% CO2 atmosphere
at 37 °C. In vivo labeling of mitochondria with a
MitoTracker (Molecular Probes) at a final concentration of 12.5 ng/ml
was performed as described previously (29).
Retroviral Infection--
BJ-E1A Ha-RasV12, BJ-E1A Ha-RasV12
MDM2, BJ-E1A Ha-RasV12 p53DN and BJ-E1A Ha-RasV12 Bcl-2 were generated
as described previously (42). Briefly, pBABE-Puro Ha-rasV12, Wzl-Neo
E1A 12S, pHygro-MaRXmdm2, pHygro-MaRX bcl2 WZLHygro DN p53 (175 H) were
used to singularly transfect the amphotropic packaging cell line LinX-A
(30). Transfection was performed by the calcium phosphate method. At
72 h after transfection, viral supernatants were collected,
filtered, supplemented with 4 mg/ml polybrene, and combined to obtain
the oncogene combinations described in the text to infect early passage
BJ cells. After infection, cells were selected with a combination of
hygromycin (50 µg/ml), puromycin (1 µg/ml), and neomycin (300 µg/ml) for 7 days. Effective infection was confirmed by Western blot analysis.
Cell Fractionation and Western Blotting--
In all the
experiments presented, cell lysates were prepared from floating death
cells and adherent cells harvested together. For subcellular
fractionation with the Dounce homogenizer, after washing, cells were
resuspended in extraction buffer B (20 mM Hepes, pH 7.5, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1mM dithiothreitol, 250 mM
saccarose, 10 µg/ml cytochalasin B, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each
of chymostatin, leupeptin, antipain, and pepstatin), incubated for 20 min on ice, and then subjected to 40 strokes in a glass Dounce
homogenizer type B. The obtained cell lysate was centrifuged twice at
13,500 × g for 10 min. The obtained pellet was
considered as crude mitochondrial fraction, and the supernatant was
considered as crude cytosolic fraction.
For Western blotting, proteins were transferred to a 0.2-µm
pore-sized nitro-cellulose membrane (Schleicher & Schuell) using a
semidry blotting apparatus (transfer buffer: 20% methanol, 48 mM Tris, 39 mM glycine, and 0.0375% SDS).
After staining with Ponceau S, the nitro-cellulose sheets were
saturated for 1 h in Blotto-Tween 20 (50 mM Tris-HCl,
pH 7.5, 200 mM NaCl 5% non-fat dry milk, and 0.1% Tween
20) and incubated overnight at room temperature with the anti-caspase-2
(31), anti-caspase-8 (Alexis), anti-caspase-7 (Cell Signaling),
anti-calnexin (Transduction Laboratories), anti-p85 polyADP-ribosyltransferase (PARP) fragment (Promega), anti-cytochrome c (Transduction Laboratories) anti-tubulin, anti-Bid (Cell
Signaling). Blots were then rinsed three times with Blotto-Tween 20 and
incubated with peroxidase-conjugated goat anti-rabbit (KPL) or
goat anti-mouse (Euroclone) for 1 h at room temperature. Blots
were then washed three times in Blotto-Tween 20, rinsed in
phosphate-buffered saline and developed with Super Signal West Pico, as
recommended by the vendor (Pierce).
Immunofluorescence Microscopy--
For indirect
immunofluorescence microscopy, cells were fixed with 3%
paraformaldehyde in phosphate-buffered saline for 20 min at room
temperature. Fixed cells were washed with phosphate-buffered saline/0.1
M glycine, pH 7.5, and then permeabilized with 0.1% Triton-X100 in phosphate-buffered saline for 5 min. The coverslips were
treated with the anti-cytochrome c antibody (Promega),
diluted in phosphate-buffered saline, for 45 min in a moist chamber at 37 °C. They were then washed with n twice and incubated with the relative TRITC-conjugated secondary antibodies (Sigma) for 30 min at
37 °C. Cells were examined with a laser scan microscope (Leica TCS NT) equipped with a 488-534 In Vitro Proteolytic Assay--
Caspase-3 was expressed in
bacteria and purified as described previously (31) using the pQE-12
expression system (Qiagen). Caspase-2 lacking the prodomain,
caspase-2
Caspase-2 was in vitro translated with 35S label
using the TNT-coupled reticulocyte lysate system (Promega). 1 µl of in vitro translated caspase-2 was incubated with
increasing amounts of recombinant caspase-3 or caspase-2 in 15 µl of
the appropriate buffer (final volume) for 1 h at 37 °C.
Reactions were terminated by adding one volume of SDS gel loading
buffer and boiling for 3 min.
Characterization of the Apoptotic Response to TSA in MCF-7 and
MCF-7 Caspase-3 Cells--
To characterize the apoptotic response
triggered by TSA, we used MCF-7, a cell line that does not express
caspase-3 activity (31). For comparison, MCF-7 was rescued for
caspase-3 either with C3WT or with its catalytically inactive form,
C3CI. These cell lines were grown in the presence of 1 µM
TSA for 44 h. To compare the proapoptotic effect of TSA, the same
cell lines were incubated with well characterized death triggers as
indicated (Fig. 1a). TSA
efficiently induced apoptosis in MCF-7/C3WT cells with ~80% of cell
death after 44 h of treatment. In MCF-7/C3CI, the apoptotic
response was reduced to 20%, thus suggesting a critical role for
caspase-3. The same experimental system was probed with other known
apoptotic inducers. The microtubule-disrupting compound colchicine
showed only a weak induction of apoptosis with ~20% of cells having
apoptotic features. Treatment with daunorubicin (DNR), which induces
genotoxic stress (33), caused cell death in ~50% of the cells, and
again, this apoptotic response was reduced in the absence of active
caspase-3. We also evaluated the apoptotic effect of the ether lipid
1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) (34). Apoptosis was induced by the ether lipid in more than 50% of the MCF-7/C3WT cells, and this response was reduced in MCF-7/C3CI cells. In the absence of serum, no significant levels of cell death could be detected.
Processing of caspase-2, -7, and -8 in response to the different
apoptotic triggers was next investigated by immunoblotting. In
MCF-7/C3WT cells that were treated with colchicine,
ET-18-OCH3, and DNR, caspase-2 typically resulted in
processed products migrating as a doublet at 33 and 32 kDa,
respectively (Fig. 1b). On the contrary, similar treatments
in MCF-7/C3CI cells resulted in lower amounts of processed caspase-2
products, thus indicating that caspase-3 is strictly required for the
observed processing. TSA treatment induced a strong processing of
caspase-2 but, in contrast to the results with other proapoptotic
agents, significant caspase-2 processing was also detectable in
MCF-7/C3CI cells. Interestingly, although in MCF-7/C3WT cells,
caspase-2 processing could be detected as p33 and p32 cleaved forms, in
MCF-7/C3CI cells, only the p33 product was detected. To confirm that
the generation of the p33 and p32 forms of caspase-2 could result from
the proteolytic activity of different caspases, in vitro
protelytic assays using recombinant caspase-2 and caspase-3 were
performed. As shown in Fig. 1c, recombinant caspase-3
processed caspase-2 and generated both the p33 and the p32 forms,
whereas when caspase-2 was processed by itself (recombinant caspase-2),
only the p33 form was observed.
In MCF-7/C3WT cells, TSA, colchicine, ET-18-OCH3, and
DNR induced activation of caspase-8, as detected by the appearance of the processed p42/41 intermediate fragments (Fig. 1b). In
MCF-7/C3CI cells, activation of caspase-8 was barely detectable in
response to colchicine, ET-18-OCH3, and DNR. TSA treatment
in the MCF-7/C3CI cells produced a clear decrease of the pro-caspase-8
as also observed for MCF-7/C3WT cells; however, only low amounts of the
p42/41 fragments can be detected in absence of caspase-3 activity.
Caspase-7 was also processed into the typical p20 fragment in response
to TSA, colchicine, ET-18-OCH3, and DNR in MCF-7/C3WT cells. In MCF-7/C3CI cells, similarly to caspase-8, TSA induces a clear
decrease in pro-caspase-7, but the amount of the p20 processing products was strongly reduced.
The same extracts were also probed for calnexin, a
caspase-3-dependent substrate and PARP that exhibits
caspase-3-independent processing (35). Whereas calnexin shows a
caspase-3-dependent cleavage in the presence of TSA, the
processed p85 PARP fragment was clearly observed in both MCF-7/C3WT and
C3CI cells treated with ET-18-OCH3, DNR, and TSA (Fig.
1b).
A time course analysis was performed to analyze, in more detail,
caspase-2 processing in response to TSA and to investigate the
caspase-3-independent cleavage of caspase-2. As shown in Fig. 1d, after addition of TSA, caspase-2 processing was readily
detected after 24 h in MCF-7/C3WT cells. In contrast, in the
caspase-3 mutant MCF-7/C3CI cells, processing of the p33 fragment was
detected, only about 12 h later than in MCF-7/C3WT. It is
noteworthy that PARP processing was increased in MCF-7/C3CI cells with
respect to MCF-7/C3WT cells. We also investigated the processing of the BH3-only Bcl-2 family member Bid (15). As shown in Fig. 1d, Bid processing, detected as loss of the p22 kDa non-cleaved Bid version, was induced by TSA, but the similar results observed in
MCF-7/C3CI and in MCF-7/C3WT cells indicated that this cleavage was
caspase-3-independent.
Caspase-9 Is Required for Cell Death, Caspase-2, -7, and -8 Activation, and PARP Processing in Response to TSA--
To further
study the mechanism by which TSA induces apoptosis, we used MCF-7 cells
expressing a dominant negative mutant form of caspase-9, MCF-7/C9DN
(36). This mutant prevents the activation of caspase-9 but not the
preceding steps of apoptosis by competing with endogenous caspase-9 for
binding to APAF-1 (37).
Treatment of control MCF-7/NEO cells with TSA induced cell death in
~20 and ~35% of cells after 48 and 60 h, respectively. Cell death was clearly reduced in MCF-7/C9DN cells with residual 5% of
death cells after 48 h and 10% of death cells after 60 h of
TSA (Fig. 2a).
As shown in Fig. 2b, processing of caspase-2, -7, -8, and
PARP in response to TSA treatment was strictly dependent on caspase-9. In fact, cleavage of caspase-2, -7, and -8 was undetectable in MCF-7/C9DN cells analyzed after 24 or 48 h of TSA treatment,
whereas caspase-2 p33 processing, caspase-8 p42/p41 processing,
caspase-7 p20 processing, and PARP p85 fragment were readily observed
in the MCF-7/NEO cells.
To confirm the critical role of caspase-9 during TSA-induced cell
death, a different caspase-9 defective cell line was analyzed. TSA
sensitivity of IMR90 fibroblasts stably co-expressing the E1A oncogene
together with a dominant negative form of caspase-9 IMR90-E1A/C9DN was
compared with that of parental IMR90 cells expressing E1A alone.
TSA response was also mediated by caspase 9 in IMR90-E1A fibroblasts
(Fig. 2c), and as observed in MCF-7/C9DN cells, no
processing of caspase-2, -7, -8, and PARP was detected in
IMR90-E1A/C9DN fibroblasts after TSA treatment (Fig.
2d).
Bcl-2 Inhibits TSA-mediated Cytochrome c Release and
Apoptosis--
During stress-induced apoptosis, cytochrome
c released from mitochondria binds to Apaf-1, and this
promotes apoptosome formation and subsequent caspase-9 activation (9,
12). Having demonstrated that caspase-9 is required to transduce the
apoptotic signal triggered by TSA, we next investigated whether TSA
promotes cytochrome c release from the mitochondria. As
shown in Fig. 3a, cytochrome c can be detected in the cytosolic fraction of MCF-7/NEO
cells cultured for 36 h in the presence of TSA, and it was not
present in the cytosolic fraction of untreated MCF-7/NEO cells.
Cytochrome c localization was also analyzed by
immunofluorescence. As shown in Fig. 3b, TSA treatment
causes alterations in mitochondrial morphology and redistribution of
cytochrome c in the cytoplasm.
On the whole, our data support the notion that TSA induces cytochrome
c release that in turn leads to a
caspase-9-dependent apoptosis. We next wanted to evaluate
the effect of Bcl-2 on TSA-mediated cell death. Previous studies
suggested that Bcl-2 can inhibit cytochrome c release and
subsequent apoptosis in cells that were treated with HDIs (22, 23).
Consistently, we observed that Bcl-2 overexpression inhibited
TSA-mediated cell death in IMR90-E1A/NEO cells (Fig. 3c).
Since Bcl-2 also antagonized cytochrome c release in
IMR90-E1A/C9DN cells (Fig. 3d), we can conclude that Bcl-2 inhibition is upstream of caspase-9. Under the same conditions, placental alkaline phosphatase (PLAP), used as a control, was unable to
block cytochrome c release from the mitochondria.
E1A Increases Susceptibility to SAHA- and TSA-induced
Apoptosis--
Different studies indicate that HDIs show selective
toxicity for cancer cells (3, 4). We analyzed the apoptotic response to
TSA and SAHA in primary IMR90 human fibroblasts as compared with IMR90
cells expressing the E1A oncogene. As an additional control, IMR90
co-expressing E1A and caspase-9 DN were employed. As revealed by the
trypan blue exclusion assays, SAHA and TSA efficiently induced cell
death in IMR90-E1A/NEO cells, whereas IMR90 parental cells showed a
reduced sensitivity to HDI-induced cell death (Fig.
4a), thus supporting the
notion of a differential sensitivity of transformed cells to HDIs. The
critical role of caspase-9 in HDI-induced cell death was further
supported by the observation of an impaired SAHA-induced apoptotic
response in IMR90-E1A cells following expression of DN caspase-9.
Consistently, caspase-2 and PARP processing in response to TSA or SAHA
was observed only in IMR90 cells expressing E1A but not in primary
IMR90 fibroblasts. Of note, E1A expression in IMR90 fibroblasts
resulted in significant higher caspase-2 levels. It has been recently
shown that E1A can coordinate the up-regulation of multiple caspases,
probably as a consequence of an unrestrained E2F activity (38). Thus
changes in caspases levels are likely to contribute to the increased
apoptotic susceptibility observed in E1A transformed cells.
Bid Processing and Translocation to the Mitochondria Require
Caspase-9 Activity--
Bid is a proapoptotic member of the Bcl-2
family of proteins that contains only a BH3 domain (15). Bid is a
caspase-8 substrate that, once processed, translocates to the
mitochondria and potently induces cytochrome c release. It
has been reported that Bid processing in SAHA-treated CEM cells occurs
even in the presence of the pancaspase inhibitor zVAD-fmk (23). We
observed that Bid processing was detectable also in absence of
caspase-3 (Fig. 1c); therefore, we wanted to investigate
whether caspase-9 was required for Bid cleavage in response to HDIs.
Immunoblotting analysis showed that during TSA- or SAHA-induced
apoptosis, caspase-9 was required to induce Bid processing, as revealed
by the disappearance of the uncleaved Bid product (Fig.
5a). Again, caspase-2
processing was strictly correlated to caspase-9 activity. Tubulin was
used as loading control.
Translocation of Bid to the mitochondria was then analyzed in
vivo by time-lapse analysis. Time lapse assays allow analysis on a
single cell basis followed in real time, and the timely appearance of
cellular shrinkage/blebbing in relation to Bid-GFP translocation to the
mitochondria could be monitored. The analysis was performed in both
IMR90-E1A/NEO and IMR90-E1A/C9DN cells. As indicated in Fig.
5b, cells were incubated for 22 h with TSA and then
microinjected with pEGFP-N1-Bid plasmid. 2 h after
injection, cells were incubated with mitotracker (29) to visualize
mitochondria, and time frames were collected every 5 min for 24 h.
Selected frames of representative experiments at the indicated times
are shown in Fig. 5c. Bid-GFP was initially detected as a
diffuse signal throughout the cell body. In IMR90-E1A/NEO cells, TSA
induced cell retraction, leading to cell collapse as early as 4.30 h after microinjection, whereas the translocation of Bid-GFP to the
mitochondria was detectable only later (Fig. 5c,
arrowheads). Neither cell collapsing, blebbing, nor
translocation of Bid-GFP to the mitochondria could be detected in
untreated cells under the same time course conditions (data not shown).
Consistent with the results presented above in IMR90-E1A-expressing caspase-9 DN cells, TSA was unable to induce cell death under the same
time frame, and Bid-GFP was still detected as diffuse staining
throughout the cell body as late as 21 h from microinjection. Histograms presented (Fig. 5d) summarize the results
obtained from various experiments where each position along the
x axis represents a single cell.
The Apototic Susceptibility to TSA and SAHA Is Dependent on
p53--
p53 tumor suppressor is the most mutated gene in cancer
cells, and it regulates cell cycle arrest or apoptosis in response to
pathological insults such as DNA damage or expression of mitogenic oncogenes (39, 40). As a consequence, inactivation of p53 can promote
oncogenic transformation and resistance to many anticancer treatments
(41). Thus defining the role of p53 during HDI-triggered apoptotic
response is key for an optimal use of these drugs in clinics. Previous
works have attempted to address this issue by using human cancer cell
lines differing in the presence/absence of p53 gene alterations
(22-24). It is clear that since cancer cell lines may have accumulated
numerous additional genetic alterations, interpretation of the results
is often difficult. Taking into account these caveats, we sought to
investigate the role of p53 in HDIs apoptotic response by using human
primary cells containing defined mutations affecting p53 function. For
this purpose, early passage normal human foreskin fibroblasts (BJ) were
transduced with E1A and Ha-RasV12 oncogenes by retroviral
infection. To selectively knock-down p53 function, these cells were
further infected with retrovirus that drive the expression of either
MDM2 or a dominant negative form of p53 (42). As a control, BJ-E1A/Ras
cells were also infected with a retrovirus encoding Bcl-2.
The trypan blue assays (Fig.
6a) show that after 24 h
of treatment with TSA or SAHA, BJ-E1A/Ras cells expressing MDM2 or
p53DN were more resistant to cell death when compared with parental BJ-E1A/Ras with functional p53. When the same analysis was performed after 48 h from treatment, cells grown in the presence of SAHA and
lacking functional p53 were still partially resistant to cell death,
whereas cells treated with TSA were almost all dead, independently from
the p53 status. Bcl-2 efficiently counteracted TSA and SAHA-induced apoptosis at both 24 and 48 h of treatment.
To confirm the role of p53 in the apoptotic response triggered by TSA
and SAHA, immunoblotting. with antibodies specific for caspase-2, Bid,
and tubulin was performed. Curiously, the p33 form of caspase-2 was
already observed in untreated BJ cells (Fig. 6b). It is
unlikely that this cleaved form is a consequence of a low level of cell
death since PARP processing was undetectable under the same conditions
(data not shown). Alternatively, it could be possible that in these
cell lines, limited proteolytic processing of caspase-2 could occur
independently from cell death or that this cleavage is a post-lysis processing.
As illustrated in Fig. 6b, the kinetics of caspase-2 and Bid
processing in BJ-E1A/Ras expressing MDM2, p53DN, or Bcl-2 treated with
TSA and SAHA was in accord with the trypan blue assays. The effect of
p53 was particularly evident in cells treated with SAHA. Even after
48 h of SAHA treatment, a large amount of uncleaved caspase-2 and
Bid could still be detected in MDM2- and p53DN-expressing cells,
whereas the same death substrates were almost completely cleaved in
BJ-E1A/Ras parental cells.
As mentioned previously, long term TSA treatment can induce cell death
also independently from p53, and accordingly, caspase-2 and Bid
proteolytic processing was almost complete, also in cells containing
p53DN or MDM2. However, a shorter TSA treatment (24 h) left a larger
amount of uncleaved caspase-2 and Bid in cells in which p53 was
functionally inactivated with respect to WT p53 parental cells.
Interestingly, acetylation of the histone H3 was more pronounced
in cells treated with TSA respect to SAHA (Fig. 6b). It is
well known that TSA is an inhibitor of HDACs at nanomolar range,
whereas SAHA is efficient at micromolar range (3). Thus the difference
in terms of apoptosis in cells treated with TSA and SAHA might reflect
the potency of the two HDIs.
The definition of the apoptotic pathway triggered by an
anti-cancer drug is important to predict the efficacy of a particular chemotherapeutic treatment including that agent. The HDIs are promising
anticancer drugs as they selectively induce differentiation and cell
death of transformed cells, and some of them are now under clinical
trials for both solid and hematological tumors (3, 4, 6).
The selectivity of HDIs toward transformed cells as compared with
untransformed normal cells has been confirmed by our work. We provide
evidence that a single oncogenic lesion, such as EIA expression,
renders IMR90 fibroblasts highly susceptible to cell death when grown
in the presence of SAHA or TSA. We have also confirmed that
mitochondria play a central role during HDI-mediated apoptotic response
(5, 23, 24, 26). In addition, our data demonstrate that the initiator
caspase-9 is critical for this cell death pathway. In IMR90-E1A and in
MCF-7 cells, abrogation of caspase-9 activity suppressed apoptosis
in response to TSA or SAHA and blocked processing of PARP, caspase-2,
-7, and -8.
TSA- and SAHA-induced processing of Bid and subsequent translocation to
mitochondria were also dependent on caspase-9. These data suggest that
Bid could play a role in the amplification loop rather than during the
initial phase of the process in HDI-induced cell death. A late function
of Bid is supported by the time-lapse analysis. Translocation of Bid to
mitochondria was observed in vivo during TSA-induced cell
death after 46.17 (±25.01) min from the appearance of the first signs
of cell death (cell shrinkage and membrane blebbing). A previous study
suggested that a caspase not efficiently inhibited by zVAD-fmk could be
responsible for Bid cleavage in response to SAHA (23). The current
study supports the notion that this caspase is caspase-9 or a caspase
regulated by caspase-9.
Our findings do not exclude a role for the initiator caspase-2 and -8 upstream of caspase-9 during HDI-induced cell death. We used proenzyme
cleavage as marker of caspase-2 and -8 activation. However, regulative
caspases might also be activated in the absence of proteolytic
processing (44). Recent studies have showed that caspase-2
can be activated independently from proteolytic processing (45), and
therefore, further investigations are needed to assess whether
caspase-2 plays a role as regulator of the mitochondrial integrity (28,
46-48), upstream of caspase-9 during apoptosis induced by HDIs.
Concerning caspase-8, a critical role of this initiator caspase during
HDI-induced cell death should be excluded since it has been reported
that its inhibition, through expression of the cowpox virus protein
CrmA, did not affect SAHA-induced cell death (23).
We demonstrate that p53 is essential for an efficient apoptotic
response to TSA and SAHA treatments. The role of p53 has been investigated by using primary human fibroblasts transformed with E1A
and Ha-Ras-V12 where p53 was inactivated by ectopic expression MDM2 or
p53DN mutant (42). Our data are in disagreement with a previous report,
which suggested the existence of a p53-independent apoptotic pathway in
response to SAHA (23). Indeed, although we find that p53 does affect
the rate of HDI-induced cell death, prolonged treatments with these
inhibitors can also induce a p53-independent response. It is thus
likely that such a delayed p53-independent response was the one
described previously (23).
Moreover, it has also been reported that conditional expression of p53
failed to modify the apoptotic response to SAHA in U937 cells (22).
U937 cells do not express p73 (51), and it is known that the p53 family
members p63 and p73 can modulate the p53 apoptotic response (49, 50).
In fact, the combined loss of p63 and p73 results in the failure of
cells containing functional p53 to undergo apoptosis in response to DNA
damage (50). Therefore, the absence of p73 expression might account for
the inability of these cells to mount a p53-dependent
apoptosis in response to SAHA (22).
A differential behavior was noted for TSA- and SAHA-induced apoptosis
relative to the rate of the dependence from p53. Cells lacking
functional p53 were still resistant to cell death after long term
treatments with SAHA (48 h), whereas apoptosis was induced in the same cells after long term treatments with TSA. One could postulate that since TSA is an inhibitor of HDACs at nanomolar range,
whereas SAHA is efficient at micromolar range (3), this difference
could mirror the potency of the two HDIs. In fact, we observed that
histone H3 acetylation was more pronounced in cells treated with
TSA.
Several models could be proposed for the relationship
between HDIs and p53. It has been reported that TSA up-regulates p53 in
endothelial cells (20), whereas HDAC1 can regulate p53 deacetylation, thereby reducing its transcriptional activity by targeting it for
degradation (19, 21). Therefore, TSA and SAHA might activate p53, thus
inducing the expression of p53 target genes involved in apoptosis.
Array analysis of gene expression in our cellular system will allow us
to better define the mechanism through which p53 regulates the
apoptotic susceptibility to HDIs.
Interestingly, production of reactive oxygen species is crucial for
cell death induced by SAHA (23, 25), and transcription of redox-related
genes, formation of reactive oxigen species, and oxidative degradation
of mitochondria components have been suggested to be critical for a
p53-dependent apoptosis (43). As schematized in Fig.
7, we propose a model where the pattern of genes affected by TSA and SAHA can be influenced by the presence of
p53 (directly or indirectly), and these genes can regulate the rate of
cell death (apoptotic threshold).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
argon laser and a 633
helium-neon laser.
-1-152, was expressed as glutathione
S-transferase fusion protein in bacteria. The activity of
purified recombinant caspase-2 was monitored by measuring the Km values toward the pentapeptide Ac-VDVAD-pNA (32). Assays of chromogenic substrate cleavage contained 50 mM
Hepes, pH 7.2, 50 mM NaCl, 0.1% CHAPS, 10 mM
EDTA, pH 8.0, 5% glycerol, 10 mM dithiothreitol.
Enzyme-catalyzed release of p-nitroanilide was monitored at
405 nm in a microtiter plate reader (Bio-Tek).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
TSA-induced caspases
processing and apoptotic cell death in MCF-7/C3WT and MCF-7/C3CI
cells. As shown in a, MCF-7/C3WT and MCF-7/C3CI cells
were treated with the indicated apoptotic stimuli, and the appearance
of apoptosis was scored with trypan blue staining as indicated under
"Experimental Procedures." b, processing of caspases in
MCF-7/C3WT and MCF-7/C3CI cells treated with different apoptotic
insults. Equal amounts of MCF-7/C3WT and MCF-7/C3CI cell lysates were
subjected to SDS-PAGE electrophoresis. Immunoblots were performed using
the indicated antibody. c, in vitro proteolytic
processing of caspase-2. [35S]methionine-labeled in
vitro translated caspase-2 was incubated for 1 h at 37 °C
with increasing amounts of recombinant caspase-2 and caspase-3.
d, time course analysis of caspase-2, Bid, and PARP
processing in MCF-7/C3WT and MCF-7/C3CI cells treated with TSA. Equal
amounts of MCF-7/C3WT and MCF-7/C3CI cell lysates at the different time
points were subjected to SDS-PAGE electrophoresis. Immunoblots were
performed using the indicated antibody.
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Fig. 2.
TSA-induced caspases processing and cell
death require caspase-9. As shown in a, MCF-7/Casp9-DN
and MCF-7/NEO cells, as indicated, were treated with TSA for the
indicated time points, and the appearance of apoptosis was scored by
trypan blue staining as indicated under "Experimental Procedures."
b, processing of caspases in MCF-7/Casp9-DN and MCF-7/NEO
cells grown for the indicated time in the presence of TSA. Equal
amounts of MCF-7/C3WT and MCF-7/C3CI cell lysates were subjected to
SDS-PAGE electrophoresis. Immunoblots were performed using the
indicated antibodies. As shown in c, IMR90-EIA/Casp9-DN and
IMR90-E1A/NEO cells were treated with TSA for the indicated time
points, and the appearance of apoptosis was scored with trypan blue
staining as indicated under "Experimental Procedures."
d, processing of caspases in IMR90-E1A/Casp9-DN and
IMR90-E1A/NEO cells grown for the indicated time in the presence of
TSA. Equal amounts of IMR90-E1A/Casp9-DN and IMR90-E1A/NEO cell lysates
were subjected to SDS-PAGE electrophoresis. Immunoblots were performed
using the indicated antibodies.
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Fig. 3.
TSA-induced cell death triggers cytochrome
c (Cyt. c) release from mitochondria,
which is counteracted by Bcl-2. a, release of cytochrome
c from mitochondria in cells treated with TSA. Crude
mitochondrial (P) and cytosolic (C) fractions
were obtained from MCF-7/NEO cells using Dounce homogenizer as
described under "Experimental Procedures." Protein samples were
prepared for Western blotting, and membranes were probed with the
anti-cytochrome c antibody. As shown in b,
MCF-7/NEO cells were treated or not (untreated) with TSA,
and after 36 h, immunofluorescence assays were performed using
anti-cytochrome c antibody. As shown in c,
IMR90-E1A/NEO cells were co-transfected with the indicated constructs
and pEGFP-N1 as a reporter. The appearance of the apoptotic cells
was scored after 20 h from transfection. Cells showing a collapsed
morphology and presenting extensive membrane blebbing were scored as
apoptotic. Data represent arithmetic means ± S.D. of four
independent experiments. As shown in d, IMR90-E1A/Casp-9 DN
cells were co-transfected with the indicated constructs and pEGFP-N1 as
a reporter. After the indicated times, from TSA treatment, cells were
fixed, and an immunofluorescence assay was performed using
anti-cytochrome c antibody. Cells co-expressing GFP and
Bcl-2 or GFP and human placental alkaline phosphatase were scored for
cytochrome c release from mitochondria. Data represent
arithmetic means ± S.D. of three independent experiments.
View larger version (36K):
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Fig. 4.
E1A increases susceptibility to SAHA- and
TSA-induced apoptosis. As shown in a, IMR90,
IMR90-E1A/NEO, and IMR90-EIA/Casp9-DN cells were treated with TSA or
SAHA for the indicated time points, and the appearance of apoptosis was
scored with trypan blue staining as indicated under "Experimental
Procedures." b and c, processing of caspases in
IMR90, IMR90-E1A/NEO, and IMR90-EIA/Casp9-DN cells grown for
the indicated time in the presence of TSA (b) or SAHA
(c). Equal amounts of IMR90, IMR90-E1A/NEO, and
IMR90-EIA/Casp9-DN cell lysates were subjected to SDS-PAGE
electrophoresis. Immunoblots were performed using the indicated
antibodies.
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Fig. 5.
Bid processing and translocation to
mitochondria require caspase-9 activity. a, processing of
Bid in IMR90-E1A/NEO and IMR90-EIA/Casp9-DN cells grown for the
indicated time in the presence of TSA or SAHA. Equal amounts of
IMR90-E1A/NEO and IMR90-EIA/Casp9-DN cell lysates were subjected to
SDS-PAGE electrophoresis. Immunoblots were performed using the
indicated antibodies. As shown in b, IMR90-E1A/NEO and
IMR90-E1A/Casp9-DN cells 24 h after seeding were treated with TSA,
and after 22 h, they were microinjected with pEGFP-N1-Bid (5 ng/µl). 2 h later, cells were subjected to time-lapse analysis
for 24 h. c, time-lapse images of representative
IMR90-E1A/NEO and IMR90-E1A/Casp9-DN cells treated with TSA and double-stained for
Bid-GFP and mitochondria. Arrowheads indicate translocation
of Bid-GFP to mitochondria. d, time-lapse sequences of
IMR90-E1A/NEO and IMR90-E1A/Casp9-DN cells overexpressing Bid-GFP and
treated with TSA. Each position along the x axis represents
a single cell. g marks the appearance of the cell shrinkage
and membrane blebbing; marks Bid translocation to mitochondria.
Cells that at the end of the analysis did not showed apoptotic features
were scored as non-apoptotic (u).
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Fig. 6.
The apoptotic susceptibility to TSA and SAHA
is dependent on p53. As shown in a, BJ cells expressing
E1A and Ha-RasV12, E1A Ha-RasV12 and MDM2, E1A Ha-RasV12 and p53DN, or
E1A Ha-RasV12 and Bcl-2 were treated with TSA or SAHA for the indicated
time points, and the appearance of apoptosis was scored with trypan
blue staining as indicated under "Experimental Procedures." As
shown in b, processing of caspase-2 and Bid in BJ cells
expressing E1A and Ha-RasV12, E1A Ha-RasV12 and MDM2, E1A Ha-RasV12 and
p53DN, or E1A Ha-RasV12 and Bcl-2 were treated with TSA or SAHA for the
indicated time points. Equal amounts of cellular lysates were subjected
to SDS-PAGE electrophoresis. Immunoblots were performed using the
indicated antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
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Fig. 7.
The critical elements of the apoptotic
pathway triggered by HDIs TSA and SAHA.
In conclusion, our findings support the notion that HDI-induced
apoptosis requires caspase-9 activation through the release of
cytochrome c in a Bcl-2-dependent and
p53-sensitive manner. This pathway seems to be common to other
anticancer-treatments; however, one difference can be noted when TSA is
compared with the DNA-damaging agent daunorubicin in cells lacking
caspase-3 activity. Bid, PARP, caspase-2, and -8 processing can still
occur in MCF-7/C3CI cells treated with TSA. This suggests that HDIs could effect the rate of caspase activation even when the amplification loop, which is largely based on caspase-3, is defective. Definition of
the molecular events that permit overcoming the caspase-3 defect is an
interesting question that requires further work.
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ACKNOWLEDGEMENTS |
---|
We are grateful to E. Aleo for helping in some experiments, to Y. Lazebnik, for MCF-7/Casp-9 DN cells and to F. Demarchi for anti-acetyl-histone H3 antibody. We also thank G. Del Sal and C. Kuhne for carefully reading the manuscript and for the helpful suggestions.
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FOOTNOTES |
---|
* This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) and CNR (Consiglio Nazionale Ricerche) Agenzia 2000 to C. B. and AIRC to R. M.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ A FIRC (Fondazione Italiana per la Ricerca sul Cancro) fellow.
To whom correspondence should be addressed. Tel.:
0432-494382; Fax: 0432-494301; E-mail:
cbrancolini@makek.dstb.uniud.it.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M213093200
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ABBREVIATIONS |
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The abbreviations used are: HDAC, histone deacetylase; WT, wild type; CI, catalytic-inactive; C3CI, caspase-3 catalytic inactive; C3WT, caspase-3 WT; C9DN, caspase-9 dominant negative; DNR, daunorubicin; ET-18-OCH3, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine; HDI, histone deacetylase inhibitor; GFP, green fluorescent protein; p53DN, p53 dominant negative; PARP, polyADP-ribosyltransferase; SAHA, suberoylanilide hydroxamic acid; TSA, trichostatin-A; zVAD-fmk, z-Val-Ala-Asp-fluoromethylketone; NEO, neomycin; TRITC, tetramethylrhodamine isothiocyanate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; E1A, adenovirus early region 1A.
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