From the Departments of Molecular Pathology and
§ Clinical Investigation, the University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 and the
¶ Department of Human Genetics, Memorial Sloan-Kettering Cancer
Center, New York, New York 10021
Received for publication, November 20, 2002, and in revised form, January 10, 2003
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ABSTRACT |
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The promyelocytic leukemia protein (PML) is a
growth/tumor suppressor essential for induction of apoptosis by
diverse apoptotic stimuli. The mechanism by which PML regulates cell
death remains unclear. In this study we found that ectopic expression
of PML potentiates cell death by apoptosis in the tumor necrosis factor The promyelocytic leukemia gene
(PML)1 was
initially identified through its fusion to retinoic acid receptor The small ubiquitin-like protein SUMO-1 modifies PML at three lysine
residues (12). This modification appears to be essential for the
integrity and function of the PML NBs. Several reports (13) documented
that modification of PML by SUMO-1 is required for the formation of PML
NBs. A PML mutant lacking the SUMO-1 sites did not form PML NBs.
Reintroduction of the wild-type PML but not the SUMO-1 mutant PML into
the PML Apoptosis is a genetically controlled suicide process consisting of two
phases. The first phase is characterized by a commitment to cell death;
the second phase, an execution phase, is characterized by membrane
inversion, exposures of phosphatidylserine residues, blebbing,
chromatin condensation, and DNA fragmentation. Two major apoptotic pathways, the mitochondria-dependent pathway and the death receptor-mediated pathway, have been well documented (18-20). The activation of an apoptotic pathway does not necessarily result in
cell death because nuclear factor NF- Our results show that PML induces PCD by a death receptor-mediated
apoptotic pathway and sensitizes cells to apoptosis upon treatment with
TNF Plasmids Construction--
The full-length PMLIV cDNA was
obtained from Professor Pierre Chambon (Institut de Chimie Biologique,
Strasbourg Cedex, France). The inducible expression plasmids pMEP4/PML
and pMEP4/PML(1-555) were constructed by subcloning the PML cDNA
or PML(1-555) into the NotI/XhoI sites of the
pMEP4 vector (Invitrogen). The plasmid pMEP4/PML(97-633) was generated
by cloning the PML(97-633) DNA fragment from pCDNA3.1/PML(97-633)
into the HindIII/XhoI site. The plasmids
pMEP4/HA-p35, pMEP4/HA-CrmA, pMEP4/Bcl-2, and pMEP4/c-FLIPs were
constructed by subcloning the full-length cDNAs amplified by PCR
into the pMEP4 vector. The expression plasmids pCDNA3/p65 and
pMEP4/p65 were created by subcloning the full-length cDNA of
p65/RelA into the BamHI/XhoI sites of pCDNA3
and the HindIII/XhoI sites of pMEP4.
pCMV/HA-NLS-p65 and pCMV/HA-NLS-LacZ were generated by cloning RelA/p65
cDNA and LacZ cDNA, respectively, into the BamHI/XbaI sites of pCMV2N3T. The
pCDNA3.1/PML(97-633) was created by cloning the
AvrII/EcoRI DNA fragment into the
BamHI and EcoRI sites of pCDNA3.1HisA. The
NF- Cell Cultures and Reagents--
The U2OS, 293T, SiHa, Saos2,
PML+/+ MEF, and PML Transfection and Luciferase Reporter Assay--
Cells were
cultured to semiconfluence and transfected with the expression plasmids
using FuGENE 6 transfection reagent (Roche Diagnostics). For
transfection into MEFs, the Effectene reagent (Qiagen, Valencia, CA)
was used. Luciferase activity was determined using the luciferase
reporter assay according to the manufacturer's protocol (Promega
Corp., Madison, WI).
Generation of Stable Cell Lines--
U2OS cells were transfected
with each of the plasmids: pMEP4 (negative control), pMEP4/PML,
pMEP4/HA-CrmA, pMEP4/HA-p35, pMEP4/c-FLIP, pMEP4/p65, and pMEP4/Bcl-2
with FuGENE 6 (Roche Diagnostics) and then selected with hygromycin
(200 µg/ml) for 10 days to establish the stable clones pMEP4/U2OS,
PML/U2OS, CrmA/U2OS, p35/U2OS, c-FLIP/U2OS, and Bcl-2/U2OS,
respectively. Inducible expression levels of the respective proteins in
each stable cell line were determined by induction with
CdSO4 (5 µM) for 20 h followed by
immunofluorescent staining and Western blot analysis.
Cell Death Analysis--
Cell death was examined by the cell
death enzyme-linked immunosorbent assay (ELISA) according to the
manufacturer's protocol (Roche Diagnostics) or by trypan blue
exclusion assay. The TUNEL assay was also performed to determine cell
death according to the manufacturer's protocol (Promega Corp.).
Colony Forming Assay--
A PML stable cell line in U2OS
(103) or the control line (pMEP4 in U2OS) was cultured in
6-well plates in DMEM containing 200 µg/ml hygromycin. After 8 h, 5 µM CdSO4 or phosphate-buffered saline (PBS) was
added, and the culture was continued for 10 days.
Subcellular Fractionation of Cytoplasmic and Nuclear
Proteins--
Cytoplasmic protein was prepared by the digitonin
extraction method. Cultured cells were washed twice with cold PBS and
resuspended in ice-cold digitonin extraction buffer (10 mM
PIPES, pH 6.8, 0.015% (w/v) digitonin, 300 mM NaCl, 3 mM MgCl2, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride). Cells were permeabilized
for 10 min and assessed by trypan blue exclusion assay and then
centrifuged for 5 min (480 × g) at 4 °C. The
supernatant contained the cytoplasmic protein. The digitonin-insoluble
pellet was resuspended in ice-cold extraction buffer (10 mM
PIPES, pH 7.4, 0.5% (v/v) Triton X-100, 300 mM sucrose,
100 mM NaCl, 3 mM MgCl2, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride)
and incubated on ice for 30 min. After centrifugation for 10 min at
5,000 × g, the nuclei were resuspended in nuclear extraction buffer (50 mM PIPES, pH 7.5, 400 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton
X-100, 0.5% (v/v) Nonidet P-40, 10% (v/v) glycerol). The nuclear
mixture was incubated for 30 min on ice and then centrifuged for 5 min
(6,780 × g) at 4 °C. The supernatant contained the
nuclear proteins.
Immunofluorescence Staining and Confocal
Microscopy--
Immunofluorescence staining was performed as described
in our previous report (6). The endogenous colocalization of p65 and
PML was determined by double-color immunofluorescence staining. Briefly, U2OS and SiHa cells were pretreated with leptomycin (5 ng/ml)
and then treated with TNF Immunoprecipitation--
Immunoprecipitation was performed as
described in our previous report (26). For coimmunoprecipitation of the
endogenous PML and p65, Jurkat cells were treated with IFN Electrophoretic Mobility Shift Assay--
The in
vitro translated PML or p65 proteins were synthesized by the
TNT-coupled wheat germ translated system (Promega Corp.). Nuclear extracts were prepared from U2OS stable cell lines or cells
treated for 1 h with TNF (20 ng/ml). The NF- PML Sensitizes Cells to Tumor Necrosis Factor
U2OS is resistant to low dose treatment with TNF
Our results demonstrated a dramatic synergistic effect between PML and
TNF
Bcl-2, Bcl-xL, and Bax are important antiapoptotic or
proapoptotic proteins that control the
mitochondria-dependent apoptotic pathway (28-32). We
evaluated whether PML regulates the expression of these proteins in
U2OS cells. Our results showed that ectopic expression of PML had no
effect on these proteins (data not shown). This result is in agreement
with a previous report (10) that deletion of the PML gene
did not affect the expression of the Bcl2 family of proteins.
Activation of Initiator/Effector Caspases and CAD Is Associated
with PML/TNF
To determine whether CAD is activated in PML-induced and
PML/TNF PML Induces Cell Death through the Death Receptor-mediated
Pathway--
The binding of TNF
Death receptor-mediated apoptosis is a very complex process and may
involve the mitochondria-dependent pathway in most cells. Recently, several viral and cellular inhibitors of apoptosis have been
found that block or halt apoptotic signaling at defined points of the
apoptotic pathways. Some examples are as follows: CrmA (the product of
cowpox virus cytokine response modifier A) (37), which is a powerful
specific inhibitor of caspase-8; p35 (the 35-kDa protein of baculovirus
AcMNPV) (38), which has a broad specificity but is a less
powerful inhibitor of caspases; c-FLIP (the cellular FLICE-inhibitory
protein) (39), a specific inhibitor of caspase-8; and Bcl-2, a cellular
inhibitor of the mitochondria-dependent apoptotic pathway
that acts by preventing cytochrome c release. To determine
the apoptotic pathways initiated by PML/TNF
Stable clones of U2OS cells that conditionally expressed CrmA
(CrmA/U2OS), p35 (p35/U2OS), c-FLIP (c-FLIP/U2OS), and Bcl-2 (Bcl-2/U2OS) were established for this study. CrmA and p35
substantially suppressed PML/TNF PML Sensitizes TNF
To determine whether PML attenuates signaling of NF-
We next evaluated whether PML is a transcriptional repressor of NF- In Vivo Association of PML and RelA/p65--
To investigate
whether PML and RelA/p65 are associated in vivo, we first
performed coimmunoprecipitation experiments using total cell extracts
isolated from cells cotransfected with PML and RelA expression
plasmids. PML was coimmunoprecipitated and associated with RelA
in vivo (Fig. 6a).
Our study further showed that the endogenous RelA and PML were
coimmunoprecipitated from the nuclear extracts isolated from cells
induced by interferon-
Because PML assembles NB by recruiting other factors in the cells, our
results suggest that PML may recruit RelA into the PML NB. To evaluate
this possibility, we performed immunofluorescent staining and confocal
microscopy of U2OS cells cotransfected with the expression plasmids of
PML or PML mutant and RelA. This study demonstrated that RelA was
indeed recruited to the PML NB in vivo in the cotransfection
experiment (Fig. 6c). The mutant PML(1-555), however,
cannot relocate RelA into the PML NB, and the negative control LacZ was
not recruited into PML NB indicating that RelA/p65 targeting into the
PML NB should be specific. These results support the idea that PML and
RelA may be associated in the PML NB in vivo and that the C
terminus of PML is required for such association. Since RelA/p65 mainly
localizes to the cytoplasm before treatment with TNF The C Terminus of PML Is Indispensable for Inhibition of
NF- RelA Overexpression Blocked PML/TNF Loss of PML Function Renders Cells Resistant to TNF
To provide further support of this finding, we investigated how PML
expression affected sensitivity to TNF This study shows that PML sensitizes cells to TNF NF- It is clear that c-FLIP specifically inhibits caspase 8, an upstream
initiator of the TNF-induced death receptor-mediated apoptosis. Recent
findings (46) demonstrated that c-FLIP is a target gene of NF- (TNF
)-resistant cell line U2OS and other cell lines. Treatment with TNF
significantly sensitized these cells to apoptosis in a
p53-independent manner. PML/TNF
-induced cell death is associated with DNA fragmentation, activation of caspase-3, -7, and -8, and degradation of DNA fragmentation factor/inhibitor of CAD.
PML/TNF
-induced cell death could be blocked by the caspase-8
inhibitors CrmA and c-FLIP but not by Bcl-2. These findings indicate
that this cell death event is initiated through the death
receptor-dependent apoptosis pathway. PML is a
transcriptional repressor of NF-
B by interacting with RelA/p65 and
prevents its binding to the cognate enhancer through the C terminus.
Coimmunoprecipitation and double-color immunofluorescence staining
demonstrated that PML physically interacts with RelA/p65 in
vivo and the two proteins colocalized at the endogenous levels.
Overexpression of NF-
B rescued cell death induced by PML/TNF
.
Furthermore, PML
/
mouse embryo fibroblasts are more
resistant to TNF
-induced apoptosis. Together this study defines
a novel mechanism by which PML induces apoptosis through repression of
the NF-
B survival pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(RAR
) involved at the breakpoint of t(15,17) (q22;q12) chromosomal
translocation in acute promyelocytic leukemia (APL) (1). The PML-RAR
fusion protein created by this translocation interferes with the normal function of PML and the RAR/retinoid X receptor pathway and plays an
important role in the pathogenesis of APL (2, 3). PML is a nuclear
protein localized in discrete subnuclear compartments designated PML
nuclear bodies (NBs) or PML oncogenic domains (4). PML is a
primary target gene of interferons (IFNs) and is widely expressed in
almost all cell lines tested (5). PML is a tumor/growth suppressor that
regulates cell cycle progression and induces cell death (6-9). The
proapoptotic and growth-suppressing functions of PML were demonstrated
in vivo using cells obtained from PML knockout mice. This
study showed that PML is required for Fas- and
caspase-dependent DNA damage-induced apoptosis in
splenocytes and is essential for the induction of programmed cell death
(PCD) by Fas, tumor necrosis factor
(TNF
), ceramide, INF
,
INF
, and INF
(10). PML also induces a caspase-independent cell
death when force-expressed in rat embryo fibroblasts (11). How PML exerts its proapoptotic effects remains unknown.
/
mouse embryo fibroblasts (MEFs) led to the
reorganization of the PML NBs (14, 15). This study convincingly
demonstrated that SUMO-1 modification of PML is necessary for the NB
formation. Recent studies (16, 17) also showed that SUMO-1 modification of PML is essential for interaction and regulation of transcriptional repression function of Daxx.
B, which up-regulates antiapoptotic genes that block cell death, is also frequently activated. For example, the activation of TNF receptor results in
caspase-8 processing, leading to the induction of cell death, whereas
NF-
B activation induced by TNF inhibits cell death (21, 22).
Therefore, the sensitivity of cells to apoptotic signals depends on
both the NF-
B-mediated survival pathway and the proapoptotic pathways. Consistent with this notion, c-Myc, E1a, and E2F1, which inhibit the NF-
B-mediated signaling pathway, enhanced PCD
(23-25).
by inhibiting the NF-
B survival pathway. PML represses
NF-
B function by interfering with its binding to the NF-
B target.
A loss of PML function in PML
/
MEFs renders cells
resistant to TNF
-induced apoptosis. These findings shed new light on
the mechanism of PML function as a tumor suppressor and define a novel
mechanism by which PML induces apoptosis through repression of the
NF-
B survival pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-Luc reporter was obtained from Clontech
Laboratories, Inc. (Palo Alto, CA).
/
MEF cells were
maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. DFF antibody (06-696), HA monoclonal antibody (mAb), and caspase-8 (05-477) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Caspase-7 (66871A) and caspase-8
antibodies (66231A) were purchased from Pharmingen. Caspase-3,
cytochrome c, and Bcl-2 antibodies were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA).
(10 ng/ml) and induced with interferon
for 24 h. Immunofluorescent staining was performed using anti-PML
mAb (PG-M3, Santa Cruz Biotechnology), anti-p65 polyclonal antibody,
and secondary antibodies. Images were captured with a Zeiss laser scan
confocal microscope (LSM 5).
(3,000 units/ml) for 48 h to induce PML expression and then with TNF
(20 ng/ml) to activate p65. A total of 500 µg of nuclear protein was
used in each coimmunoprecipitation assay.
B probe was prepared by annealing the complementary oligonucleotides
(5'-AGTTGAGGGGACTTTCCCAGG), and the 3'-recessive ends were labeled with
Klenow fragment fill-in reaction. Binding reactions contained 5 µg of
nuclear extracts, 1 µg of poly(dI·dC), 1 ng of NF-
B probe
(1 × 105 cpm) in 20 µl of KCl binding buffer (10%
glycerol, 1 mM EDTA, 5 mM dithiothreitol, 20 mM Tris-HCl, pH 8.0, and 5 mM KCl). The reaction was incubated for 20 min at room temperature and then resolved
in a 5% polyacrylamide gel in Tris glycine buffer (50 mM
Tris, 0.4 M glycine, 2 mM EDTA, pH 8.5). For
competition or supershift assays, 50 ng of unlabeled probe or 1 µg of
anti-p65 mAb was added to the binding reactions, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Induced
Apoptosis--
It is well documented by animal and cell culture models
that PML is a tumor/growth suppressor. To understand further the
mechanism of the growth-suppressing function of PML, we generated
stable PML clones in U2OS, a human osteogenic sarcoma cell line. In
these cells, expression of PML is driven by the metallothionein
promoter, inducible by Cd2+ or Zn2+. U2OS cells
transfected with vector alone were used as a control. The colony growth
of these cells was significantly inhibited when PML expression was
induced by Cd2+ but not in the control cells (Fig.
1, a and b), demonstrating the
growth-suppressing property of PML in U2OS. The inducible expression
level of PML in this cell line is comparable with that in SiHa cells
after induction with IFN
(Fig. 1a).
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Fig. 1.
PML functions as a proapoptotic protein.
a, inducible expression of PML in the PML/U2OS cell line by
various concentrations of CdSO4 for 16 h. Total
proteins were isolated from cells treated with the indicated
concentration of CdSO4, and Western blot analysis was
performed using the PML polyclonal antibody. Western blotting was also
performed using total proteins isolated from SiHa and U2OS cells
treated or untreated with 1000 units/ml interferon-
(INF-
). b, inducible expression of PML
repressed colony formation of U2OS cells. PML/U2OS stable cells (5 × 103) and the negative control (pMEP4/U2OS) were spread
onto 6-well plates. After incubation for 6 days in the presence of 5 µM CdSO4 or PBS (negative control) in DMEM
containing 200 ng/ml of hygromycin, image was captured in an Alpha
Innotech Gel Documentation System. c, the TNF
receptor-mediated apoptotic pathway exists in U2OS cells. U2OS cells
(2 × 105) were seeded in a 6-well plate and cultured
for 16 h. Cells were then treated with 1 µg/ml cycloheximide
(CHX) for 2 h. TNF
(10 ng/ml) was then added, and
the cells were incubated for an additional 16 h. Percent cell
death was measured by trypan blue exclusion assay. d, PML
sensitizes TNF
-induced apoptosis in U2OS cells. PML/U2OS or
pMEP4/U2OS was induced for 8 h with 5 µM
CdSO4, and the cells were then treated or untreated with
TNF
(10 ng/ml) for 24 h. Cell death was quantified by
cell-death detection ELISA assay (Roche Diagnostics).
(27), but apoptosis
can be induced by TNF
in the presence of the protein synthesis
inhibitor cycloheximide (Fig. 1c). This indicates that the
TNF receptor-mediated apoptotic pathway is intact in this cell line. We
next investigated whether the induction of PML expression by
Cd2+ in PML/U2OS stable clones resulted in cell death. Our
results showed that the expression of PML was insufficient to induce
cell death in a significant number of cells within 24 h. We next
evaluated whether the death receptor-mediated apoptotic pathway is
utilized in PML-induced cell death. The results demonstrated that a
combination of PML and TNF
resulted in massive apoptosis (Fig.
1d). Similar results were observed when ectopic expression
of PML was achieved by infection with recombinant PML adenovirus,
Ad-PML (data not shown).
in the induction of apoptosis in U2OS cells. To examine whether
PML/TNF
-induced apoptosis is associated with DNA fragmentation, U2OS
cells were infected with Ad-PML or the control adenovirus Ad-C for
8 h and then treated with TNF
for an additional 16 h. Cell
death was examined by TUNEL assay. As expected, PML expression alone
was insufficient to induce cell death in 24 h; a combination of
PML and TNF
, however, induced a massive DNA fragmentation (Fig.
2a), suggesting that
caspase-activated DNase (CAD) was activated during PML/TNF
-induced
cell death. We next sought to determine whether similar effects could
be achieved in other cell lines. We found that PML expression
dramatically sensitized TNF
-induced apoptosis in Saos2, HT1080, and
293T cell lines (data not shown).
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Fig. 2.
DNA fragmentation, activation of caspase-3,
-7, and -8, and CAD are associated with PML/TNF -induced cell
death. a, detection of Ad-PML-induced DNA fragmentation
by TUNEL assay. U2OS cells (2 × 105) were cultured in
a 6-well plate for 12 h and then infected with Ad-C, Ad-PML, and
PBS (Mock) for 8 h. The infected cells or mock control cells were
then treated with TNF
for 16-24 h. Cell death was examined using
the Apoptosis System Fluorescence kit (Promega Corp.). Nuclear DNA was
stained with Hoechst dye. b, processing of caspase-3, -7, and -8 and DFF/ICAD in PML/TNF
-induced cell death. Total protein
samples prepared from cells treated as described in Fig. 2 legend were
resolved in 12% SDS-PAGE. Western blot analysis was performed using
the indicated specific antibodies. The same blots were reprobed with
anti-actin or
-tubulin antibody to serve as an internal control.
c, activation of caspase-8 protease in the
PML/TNF
-treated cells. Cells were infected with Ad-C or Ad-PML for
8 h and then treated with TNF
(10 ng/ml) for an additional
period of 16 h. The caspase-8 enzyme activity assay was performed
using IETD-p-nitroanilide substrate according to the
manufacturer's protocol (Clontech Laboratories,
Inc.). Results shown represent an average value of two independent
experiments.
-induced Apoptosis--
The results of our study
suggest that the death receptor signaling pathway is involved in
PML/TNF
-induced cell death. To confirm this notion, we evaluated
whether PML/TNF
-induced cell death involves the activation of
initiator/effector caspases and CAD, which are crucial players in
apoptosis for almost all cell types. It has been well documented that
upon ligand binding, TNF receptors recruit pro-caspase-8 via the
adaptor protein FADD and subsequently cleave effector caspases such as
caspase-3, -6, and -7. This activates CAD by degrading the inhibitor of
CAD (ICAD). Consequently, DNA fragmentation and apoptosis occur (33).
These active executioners also cleave other cellular substrates that are essential for cell survival and responsible for the morphologic and
biochemical features of apoptosis.
-induced cell death, we infected the U2OS cells with Ad-PML or Ad-C for 8 h. TNF
was then added for an additional 16 h. CAD activation was then detected by examining the degradation of
DFF/ICAD. Western blot analysis demonstrated that DFF/ICAD was degraded in the PML/TNF
-treated cells in 24 h (Fig. 2b).
Initiator caspase-8 and effectors caspase-3 and caspase-7 were also
activated in the PML/TNF
-treated cells (Fig. 2b). The
activation of caspase-8 by PML/TNF
was further confirmed by a
caspase-8 activity assay (Fig. 2c). Caspase-8 is an apical
enzyme that mediates the death receptor apoptotic pathway and is
capable of processing almost all other caspases (33). Our finding
supports the hypothesis that PML/TNF
-induced cell death involves the
death receptor-mediated apoptotic pathway.
with death receptors
activates caspase-8 and processes effector caspases, including
caspase-3, -6, and -7. Bcl-2 cannot rescue these cells from
TNF
-induced apoptosis in most cell lines. Active caspase-8 can also
process the proapoptotic Bcl-2 family member Bid (34) that contains
only BH3. Truncated Bid translocates to mitochondria and induces the
loss of mitochondria membrane potential (
m) and releases
cytochrome c from the mitochondrial intermembrane space to
the cytosol. The released cytochrome c binds to Apaf1 and
then recruits and processes pro-caspase-9 and initiates the downstream
caspase cascade (35). Therefore, the caspase-8-mediated pathway can use
mitochondria to activate the executioner apoptosis caspase cascade
(36). In contrast, the apoptosis initiated by the
mitochondria-dependent apoptosis pathway can be inhibited
by Bcl-2/Bcl-XL, which blocks cytochrome c
release (30).
, we assessed the ability
of these apoptosis inhibitors to block PML/TNF
-induced apoptosis.
-induced cell death within 24 h
(Fig. 3a). CrmA exerted a much
stronger inhibitory effect than did p35. Cellular FLICE-inhibitory
protein c-FLIP, which blocks TNF receptor-mediated apoptosis, also
inhibits PML/TNF
-induced cell death (Fig. 3b). In a
similar experiment, Bcl-2 did not inhibit PML/TNF
-induced cell
death, although it retained its ability to block cytochrome c release from the mitochondria (Fig. 3c). It is
interesting to note that Bcl-2 did not affect pro-caspase-8 processing.
This finding is in agreement with the previous reports (40) that showed
that Bcl-2 could not block TNF
-induced apoptosis in some types of
cells. Together, these results strongly support the idea that
PML/TNF
-induced cell death depends upon the death receptor-mediated pathway.
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Fig. 3.
PML potentiates cell death through the death
receptor-mediated pathway. a, viral inhibitors of apoptosis
inhibit PML/TNF -induced cell death. Inducible stable lines were
induced for 12 h with CdSO4 (5 µM) and then infected
with Ad-PML. After 8 h, infected cells were treated with TNF
(10 ng/ml) for 16 h. Cells were then harvested and analyzed for
cell death by ELISA as described above. b,
PML/TNF
-induced cell death can be inhibited by c-FLIPs but not
Bcl-2. Inducible and stable cell lines expressing c-FLIPs and Bcl-2
were treated as described in a before analysis of cell
death. c, Bcl-2 blocked cytochrome c released
from the mitochondria but not caspase-8 processing and cell death
induced by PML/TNF
. The indicated inducible-stable cell lines were
treated as described in a. Cell death was determined by
trypan blue exclusion assay. The cytosolic and total proteins were
extracted and resolved in a 10% SDS-PAGE. Western blot analysis of the
cytosolic protein was performed with the antibodies against cytochrome
c, caspase-8, and
-tubulin. Western blot analysis of
total proteins was performed using the rabbit anti-PML antibody.
-induced Apoptosis by Acting as a Functional
Inhibitor of NF-
B--
The TNF
signaling events activate the
proapoptotic pathway and the antiapoptotic pathways through the
activation of caspase-8 and NF-
B transactivation function,
respectively (41). NF-
B consists of two subunits (p65 and p50 or
p52) localized in the cytoplasm to form an inactive complex with an
inhibitor of NF-
B (I
B) (41). TNF
induces I
B kinase
activation that leads to phosphorylation and subsequently degradation
of I
B by proteosome. NF-
B then enters the nucleus and activates
the transcription of various target genes, including those encoding the
antiapoptotic proteins (22, 42). Therefore, it is possible that PML
sensitizes TNF
-induced cell death by interfering with NF-
B signaling.
B when cells are
treated with TNF
, we cotransfected an NF-
B-dependent luciferase reporter with the PML expression plasmid. PML dramatically repressed the transactivation of NF-
B induced by TNF
(Fig.
4a). This finding suggests
that PML is a negative regulator of the NF-
B signaling pathway.
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Fig. 4.
PML is a transcriptional repressor of RelA
(p65). a, PML inhibits activation of NF- B induced by
TNF
. U2OS cells were cotransfected with NF-
B reporter
(NF-
B)-TATA-Luc and pCDNA3/PML or pCDNA3. After treatment
with TNF
at the indicated time points, total proteins were isolated,
and luciferase activity was determined using the luciferase assay kit
(Promega). The expression plasmid pCMV/
-Gal was included in each
transfection, and
-galactosidase activity was determined to
normalize transfection efficiencies. b, PML represses the
transcriptional activation mediated by NF-
B. (NF-
B)-TATA-Luc
reporter (0.3 µg) was cotransfected with 0.6 µg of pCDNA3 or
pCDNA3/PML and the indicated amounts of pCDNA3/p65 into U2OS
cells. Luciferase activity was determined after 24 h as described
in a. c, PML inhibits transcriptional activity of
NF-
B in a dose-dependent manner. (NF-
B)-TATA-Luc or
control reporter UAS-TATA-Luc was cotransfected with pCMV/p65 (20 ng)
and an increasing concentrations of pCDNA3/PML into U2OS cells. The
pCMV/
-gal was included in all transfection assays to monitor
transfection efficiency. Luciferase activity in each assay was
determined and normalized by
-galactosidase activity after 24 h. d, PML enhances Myc-mediated transactivation of reporter
in a dose-dependent manner. The reporter plasmid
pMyc-TA-Luc was cotransfected with pcDNA3/c-myc and various
concentrations of pcDNA3/PML into U2OS cells. Luciferase activity
was determined after 24 h post-transfection as described in
a. The pCMV/
-Gal was included in each transfection assay
to normalize transfection efficiency.
B
activity. A series of cotransfection experiments was performed using
RelA (p65), PML expression plasmids, and the NF-
B reporter
construct. This study demonstrated that PML significantly repressed the
transcriptional activity of RelA (p65) (Fig. 4b). In
addition, PML inhibited RelA-mediated transcription in a
dose-dependent manner (Fig. 4c). In a similar
experiment, we found that cotransfection of PML up-regulates
c-Myc-mediated transactivation, indicating that the repression effect
of PML on p65-mediated transcription is specific (Fig. 4d).
These results suggest that NF-
B is a direct target of PML. Because
PML does not bind DNA, one possible explanation for such inhibitory
effect is that PML interacts with RelA and interferes with its binding
to the promoter of target genes. Electrophoretic mobility shift assay
demonstrated that PML significantly inhibited RelA binding to its
consensus enhancer sequence when the in vitro translated
proteins (Fig. 5a) or the
nuclear extracts were used in the assay (Fig. 5b). To
determine whether PML could stabilize I
B or affect the expression of
RelA/p65 during TNF
-triggered signaling, Western blotting was
performed using total protein isolated from PML/U2OS and pMEP4/U2OS
induced with TNF
. This study showed that PML had little effect on
the stability of I
B and expression of RelA/p65 (Fig.
5c).
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Fig. 5.
PML inhibits NF- B binding to its
cognate enhancer sequence. a, PML inhibits the binding of
NF-
B to its cognate enhancer. Electrophoretic mobility shift assay
was performed using 1 µl of in vitro translated p65 and an
increasing concentration of in vitro translated PML protein.
b, PML inhibits NF-
B activation in response to TNF
in vivo. The inducible PML or pMEP4 (vector)
stable lines in U2OS were pretreated with 5 µM
CdSO4 for 16 h and then treated with TNF
(20 ng/ml)
for 20 min. Nuclear extract were isolated, and NF-
B activation was
determined by electrophoretic mobility shift assay. c, the
effects of PML on expression of I-
B and p65/RelA. Expression of the
PML protein was induced by 5 µM CdSO4 for
16 h. Total protein was then isolated, and expression of I-
B
and p65/RelA was analyzed by Western blot analysis. SS,
supershift; NS, nonspecific.
and pretreated with TNF
(Fig.
6b). These results strongly suggest that the two proteins
are associated in vivo.
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Fig. 6.
In vivo association of PML and
RelA/p65. a, coimmunoprecipitation of PML and p65. U2OS
cells were transfected with pCMV/HA-NLS-p65 and cultured for 24 h.
Cells were then infected with Ad-PML for 16 h. Total proteins were
isolated and coimmunoprecipitated with anti-HA monoclonal antibody or
with anti-PML polyclonal antibody. The precipitated complex was
resolved in an 8% SDS-PAGE and probed with anti-PML antibody or
anti-HA mAb, respectively. b, association of endogenous PML
and RelA (p65). PML proteins expression were induced with IFN for
48 h in Jurkat T cells and then treated with TNF
for 45 min.
Nuclear proteins were extracted and coimmunoprecipitated
(Co-IP) with anti-p65 mAb (left panel) or with
anti-PML polyclonal antibody (right panel). The precipitated
complex was resolved by 8% SDS-PAGE and probed with anti-PML antibody
or anti-p65 antibody, respectively. WB, Western blot.
c, PML recruits RelA/p65 into PML NB. The pCDNA3/PML and
pCMV/HA-NLS-p65 (upper panel) or pCDNA3/PML(1-555) and
pCMV/HA-NLS-p65 (middle panel), or pCDNA3/PML and
pCMV/HA-NLS-LacZ (lower panel) expression plasmid
combinations were cotransfected into U2OS cells grown on slides for
18 h before the slides were sequentially immunostained with
anti-HA mAb and anti-mouse IgG-conjugated with rhodamine and then with
anti-PML antibody and anti-rabbit IgG conjugated with fluorescein
isothiocyanate. HA-LacZ was transfected and stained as a negative
control. d, confocal microscopic analysis of the endogenous
colocalization of p65 and PML in Siha and U2OS cells. Cells were
pretreated with leptomycin (5 ng/ml) and TNF
(10 ng/ml) and then
were induced with interferon
for 24 h. Cells were stained with
anti-PML mAb and anti-p65 polyclonal antibody followed by fluorescein
isothiocyanate-conjugated or rhodamine-conjugated secondary
antibody.
or other
stimuli, it is important to examine whether PML can also recruit the
endogenous RelA/p65 to the PML NB when RelA/p65 is translocated into
the nuclei after TNF
treatment. To evaluate this possibility, SiHa
and U2OS cells were pretreated with leptomycin B, which has been shown
to retain RelA/p65 in the nuclei, and then were treated with TNF
and
interferon. Double-color immunofluorescent staining and confocal
microscopy detected colocalization of the endogenous RelA/p65 and PML
in the PML NB (Fig. 6d) in both the SiHa and U2OS cells.
These results strongly support the idea that PML and RelA/p65 are
functionally associated in vivo.
B--
Our study demonstrated that PML functionally represses
RelA/p65 by recruiting it to the PML NB and interfering with binding of
NF-
B to its enhancer. We next attempted to identify which domain of
PML is required to repress NF-
B transactivation. By using several
PML mutants in a series of cotransfection experiments with the NF-
B
reporter construct, we found that PML mutants with a deletion of the
RING region were capable of inhibiting NF-
B transactivation, whereas
PML mutants lacking amino acids 555-633 (PML(1-555)) and 305-633
(PML(1-305)) were not. This result
indicated the C terminus of PML is essential for PML to fully inhibit
NF-
B (Fig. 7a). Interestingly, a previous report
showed that the C terminus of PML is required for interactions with p53
and relocation of p53 to the PML NB (62). To determine whether the
ability of the PML to inhibit NF-
B is required for the
proapoptotic function of PML, a stable cell line expressing
PML(1-555) was established in U2OS cells. We compared the sensitivity
to TNF
treatment of this stable cell line with that of the wild-type
PML. Our results demonstrated that the C terminus of PML is required
for sensitizing TNF
-induced apoptosis (Fig. 7b). Result
presented in Fig. 6c showed that PML(1-555) failed to
recruit p65 to the PML NBs. We next performed coimmunoprecipitation
assay to investigate whether PML(1-555) physically interacts with p65.
This study indeed demonstrated that PML(1-555) was unable to
coimmunoprecipitate p65 (Fig. 7c). Taken together, these
results demonstrated that the C terminus of PML (amino acids 556-633)
is responsible for inhibiting NF-
B transactivation, recruiting
NF-
B to the PML NB, and enhancing TNF
-induced apoptosis.
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Fig. 7.
The C terminus of PML is required for
inhibition of NF- B activity and sensitizing TNF
-induced
apoptosis. a, the C terminus of PML is required for complete
inhibition of NF-
B activity. NF-
B reporter plasmid was
cotransfected with pCDNA3/p65 and the indicated PML mutants into
U2OS cells. Luciferase activity was assayed at 24 h
post-transfection. b, the C terminus of PML is required for
sensitizing cells to TNF
-induced apoptosis. The inducible stable
cell lines expressing wild-type PML, PML(1-555), or vector (pMEP4)
were pretreated with 5 µM CdSO4 for 16 h. TNF
(10 ng/ml) was then added, and cells were cultured for an
additional 24 h. Cell death was then determined by the cell death
detection ELISA and normalized by the untreated samples. c,
PML(1-555) fails to physically interact with p65/RelA.
Coimmunoprecipitation was performed as described in Fig. 6 legend. U2OS
cells were cotransfected with pCMV/HA-NLS-p65 and pcDNA3/PML
(lane 1) or pCMV/HA-NLS-p65 and pcDNA3/PML(1-555)
(lane 2). Lane 3 shows the negative control using
mouse or rabbit IgG. Coimmunoprecipitation (IP) and Western
blotting (WB) were performed using the anti-HA monoclonal
antibody or the PML polyclonal antibody (pAb).
-induced
Apoptosis--
If PML sensitizes U2OS cells to TNF
-induced
apoptosis by inhibiting the NF-
B-mediated survival pathway as
our studies suggest, then ectopic expression of RelA/p65 should block
PML/TNF
-induced apoptosis. To test this hypothesis, a RelA-inducible
stable cell line was established in U2OS cells and was infected with
Ad-PML or Ad-C in the presence or absence of CdSO4 and then
treated with TNF
. Apoptosis was then quantified by a DNA
fragmentation assay. The result of this study demonstrated that
overexpression of RelA significantly reduced PML/TNF
-induced
apoptosis (Fig. 8). This PML/TNF
-induced cell death was associated with DFF/ICAD degradation. These results demonstrated that PML is a functional inhibitor of
RelA/p65.
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Fig. 8.
RelA expression blocked
PML/TNF -induced apoptosis. The
RelA/p65-inducible stable cell line was pretreated with 5 µM CdSO4 for 16 h to induce the
expression of RelA. Cells were then infected with Ad-C or Ad-PML for
8 h and then treated with TNF
(10 ng/ml) for 16 h. Cell
death was quantified by the cell death detection ELISA. Total proteins
were isolated from the same samples and resolved in a 10% SDS-PAGE.
Western blot analysis was performed with anti-DFF/ICAD antibody and
anti-tubulin mAb.
-induced
Apoptosis--
We next sought to examine the effects of endogenous PML
on TNF
-induced signaling events in PML+/+ and
PML
/
MEFs. A culture medium with low growth and
low survival factors was selected to sensitize the wild-type MEFs to
apoptosis in response to TNF
treatment. MEFs derived from
PML+/+ and PML
/
mice were tested for their
relative sensitivity to TNF
under similar conditions.
PML+/+ and PML
/
MEFs exhibited similar
survival rates, but the PML
/
MEFs were significantly
more resistant than the PML+/+ MEFs to TNF
-induced
apoptosis (Fig. 9a). This
experiment was repeated, and similar results were observed. This
finding implies that a loss of PML function rendered cells resistant to
TNF
-induced apoptosis.
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Fig. 9.
Loss of PML function rendered cells resistant
to TNF -induced apoptosis. a, the
effects of endogenous PML on TNF
receptor-mediated apoptosis
signaling pathway. PML+/+ MEF and PML
/
MEF
were cultured in DMEM containing 0.5% serum and then treated with
TNF
(50 ng/ml) for 48 h. Cell death was quantified by the cell
death detection ELISA. b, reintroduction of PML into
PML
/
MEF restored TNF
-mediated cell death. The MEFs
and PML
/
MEF were infected with Ad-PML and Ad-C in the
presence or absence of TNF
. Cell death was quantified as described
in a. c, promoter activity of NF-
B in
PML+/+ MEF and PML
/
MEF. The NF-
B
reporter plasmid (NF-
B)-TATA-Luc was transfected into the
PML+/+ MEF and PML
/
MEF. Cells were
cultured for 18 h and then treated with 10 ng/ml of TNF
for
4 h. Total proteins were isolated, and luciferase activity was
determined as described in Fig. 4 legend.
-mediated cell death.
PML+/+ MEFs and PML
/
MEFs were infected
with Ad-PML and the control Ad-C. The results of this study showed that
reintroduction of PML into the PML
/
MEFs restored
sensitivity to TNF
-mediated cell death (Fig. 9b). We next
examined whether TNF
increases activity of NF-
B transactivation in PML-deficient cells in a transient transfection assay. The results
demonstrated a moderate but consistent increase in reporter activity in
the PML
/
MEFs (Fig. 9c).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced
apoptosis in U2OS and several other cell lines through the death receptor-dependent apoptotic pathway. The C terminus of PML
is indispensable for the proapoptotic functions of the PML. The
proapoptotic function of PML is p53-independent, as shown by the effect
of the PML on the p53-negative Saos-2 cell line. By using the
TNF
-resistant U2OS cell line as a model, we showed that PML
sensitizes TNF
-induced cell death by activating caspase-8, -7, and
-3 and degrading DFF/ICAD. By using stable cell lines with inducible
expression of several of the inhibitors of apoptosis, our study showed
that PML/TNF
induced apoptosis through the death receptor-mediated
pathway. Overexpression of Bcl2 did not effectively inhibit cell death induced by PML/TNF
, but p35, CrmA, and c-FLIP did. This observation does not support the involvement of the mitochondria pathway. The
results of this study support the notion that PML/TNF
-induced apoptosis is mediated through the death receptor apoptotic pathway.
B activation can be found in many different types of cancers,
serving as a mechanism to prevent cancer cell death and contributing to
tumorigenesis (44). We examined whether apoptosis could be induced
through the death receptor-mediated pathway by regulating the NF-
B
survival pathway. Our results (Figs. 4 and 5) demonstrated that PML
represses the transactivation function of NF-
B by interacting with
p65/RelA and preventing it from binding to the NF-
B recognition
sequence. This result is in agreement with our previous report (45)
that demonstrated that PML repressed A-20-mediated transcription, a
target gene of NF-
B, through the NF-
B-binding site.
Coimmunoprecipitation of PML and p65/RelA was found at the endogenous
levels and supports an in vivo association between the two
proteins. This notion was further supported by the finding that PML
colocalizes with p65/RelA at the endogenous levels in both U2OS and
SiHa cells. Our results further showing that stable overexpression of
RelA inhibits PML/TNF
-induced apoptosis (Fig. 8). Together, these
studies demonstrated that PML sensitizes TNF
-induced apoptosis by
inhibiting the NF-
B survival pathway. Further support for this
conclusion was obtained using the PML knockout MEFs in a TNF
-induced
cell death assay. This study showed that PML-deficient cells are more
resistant to TNF
-induced apoptosis than are normal MEFs and that
TNF
sensitivity in these cells can be restored when PML expression
is reintroduced by adenovirus-mediated gene transfer.
B and
that restoration of FLIP in NF-
B-null cells efficiently inhibits
TNF- and FasL-induced apoptosis. This finding, together with our
results presented here, demonstrates that PML sensitizes TNF
-induced
apoptosis by inhibiting the NF-
B transactivation function.
Down-regulation of NF-
B transactivation relieves c-FLIP inhibition
of caspase-8, leading to the activation of this apoptosis initiator and
downstream effector caspases. At the same time, other target genes of
NF-
B, including several of the inhibitor of apoptotic proteins,
e.g. IAPs, will also be down-regulated, resulting in further
activation of effector caspases. Together, these events weaken the
NF-
B survival pathway and trigger apoptotic cell death (see Fig.
10). Our studies that overexpression of
Rel A and c-FLIP inhibits PML/TNF
induced apoptosis (Fig. 3b and Fig. 8) support this hypothesis.
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Fig. 10.
A schematic illustration of
PML/TNF -induced apoptosis through
inhibition of the NF-
B survival pathway.
TNF
-induced activation of upstream initiator caspase-8 further
activates several of the downstream caspases including caspase-3, -6, and -7. CADs become activated and resulted in DNA fragmentation and
eventually apoptosis. At the same time, TNF
also induced activation
of NIK which activates NF-
B by induced phosphorylation and
degradation of I-
B. NF-
B in turns activates many of the
downstream target genes including anti-apoptotic genes
cFLIP, IAPs, Bcl-XL,
and A20 and prevent cells from undergoing apoptosis. PML
sensitizes TNF
-induced apoptosis by acting as a functional inhibitor
of NF-
B.
NF-B plays a central role in host defense and inflammatory
responses, and its activity can be activated rapidly by many
proinflammatory agents, including cytokines and virus (47). NF-
B
regulates a wide variety of genes, including many of the genes encoding cytokines, chemokines, and adhesion molecules (47-49). Many of the
apoptosis inhibitory proteins, including IAPs, A1, A20,
Bcl-XL, FasL, TRAFs, and c-FLIP, are also targets of
NF-
B (48, 51). An important role of NF-
B in anti-apoptosis was
first demonstrated by Beg et al. (49), who showed that
p65/RelA-deficient mice died in the embryo from extensive liver
apoptosis at E15. NF-
B activity is normally controlled by I
B, a
cytoplasmic protein that forms an inactive complex with NF-
B and
inhibits NF-
B activity by preventing it from entering the nucleus.
Our study shows that PML, a nuclear protein, also inhibits NF-
B
activity. Thus NF-
B-mediated signaling could be regulated in both
the cytoplasm and the nucleus. In addition, TAFII 105 is a
nuclear transcriptional coactivator of NF-
B essential for induction
of antiapoptotic proteins in response to TNF
(52). A
dominant-negative TAFII 105 blocked the interaction between
NF-
B and TAFII 105 and severely reduced cell survival in
response to TNF
(53). Another IFN-inducible protein, p202 (50), was
shown to inhibit NF-
B activity in the nucleus and TNF
-induced
sensitized cell death through a mechanism similar to that utilized by
PML.
PML is involved in viral DNA replication, and it appears that the PML
NB-associated proteins are released to viral replication and
transcription domains. The two early transcribed adenovirus proteins
E4-open reading frame 3 and E1B (54, 55) are targeted to the PML NB and
trigger its dissociation from other cellular factors or the release of
some important factors that are required for viral propagation and the
prevention of apoptosis of host cells. The inhibition of the PML NB
dissociation reduces adenovirus replication, supporting the idea that
the PML NB retains cellular factors that play critical roles in viral
replication, regulation of viral transcription, and host-cell survival.
It is well documented that NF-B is frequently activated by viral
infection and plays a critical role in viral oncogenesis and the
regulation of viral gene transcription (56). Our finding that PML
represses NF-
B (p65) transcriptional activity may help explain why
the PML NB is the target of several virus proteins. The PML NB has been
shown to be the target of adenovirus, human T-cell leukemia virus type 1 (57), papillomavirus (58), hepatitis virus (59), herpes simplex virus
(60), and human cytomegalovirus (61).
Other mechanisms of PML-induced apoptosis have also been reported. A
role of PML in p53-dependent apoptosis in thymocytes has
been reported recently (62) in response to ionization radiation. This
pathway involves activation of the p53 downstream target genes
bax and p21. Another pathway of
PML-induced apoptosis was also reported recently. This pathway is
induced in response to Fas in B and T splenocytes in a p53-independent
manner through a mechanism involve the in vivo association
between PML and the proapoptotic protein Daxx (9, 63). Daxx was
originally identified as a Fas death domain binding protein (64); it
also directly interacts with PML and colocalizes in the PML NBs. Daxx
regulation of Fas-induced apoptosis required the ability of Daxx to
colocalize to the PML NBs (65). Our study here shows that
PML/TNF-induced apoptosis is a mechanism independent from the
p53 function. It is not clear at this stage whether this mechanism is
in anyway connected with the Fas/Daxx-mediated apoptotic pathway. It
has been shown that Daxx acts as a transcriptional repressor by
recruiting histone deacetylases. The SUMO-1-modified PML negatively
regulates transcriptional repression of Daxx by interacting and
sequestering Daxx to the PML NBs (17, 18). Although the detailed
mechanism of Fas/Daxx-induced apoptosis remains unclear, it is
possible that PML regulates this pathway through a direct interaction
with Daxx.
PML and its associated proteins play a critical role in the control of
cell growth, although the molecular mechanism is not clear. Recent
findings (62, 66) demonstrated that p53 was recruited to the PML NB
through a direct interaction between the core domain of p53 and the C
terminus of PML, resulting in enhanced transactivation of p53 in a
promoter-specific manner and affecting cell survival. This study
implies that the PML NB is required but alone is insufficient to
enhance p53-induced apoptosis. Although it lacks the C terminus,
PML(1-555) forms a nuclear body (67) but cannot enhance p53-mediated
cell death. Interestingly, the C terminus of PMLIV isoform is also
essential for the interaction with p65 (Fig. 6c and
7c). More important, the PML mutant lacking the C terminus
could not fully inhibit p65 activity and lost its ability to enhance
apoptosis in response to TNF. Various PML isoforms have been
found that share the same N terminus with variable C termini generated
by alternative splicing (68). We speculate that the various functions
of PML may be regulated through alternative splicing to produce various
PML isoforms with different cellular functions. Our previous study (26)
demonstrated that only a specific isoform of PML interacts with
histone deacetylases for transcriptional repression. There is
also evidence that only some PML isoforms interact with
retinoblastoma protein. It is therefore important in the future
to study how PML regulates transcription, cell growth, and apoptosis
through specific isoforms.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Mariann Crapanzano for editing and critical reading of the manuscript. The DNA sequencing and the confocal microscopy facilities were supported by NCI Research Grant CA-16672 from the National Institutes of Health.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant CA 55577 from the National Institutes of Health (to K.-S. C.).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.
To whom correspondence should be addressed: Dept. of Molecular
Pathology, the University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Unit 89, Houston, TX 77030. Tel.:
713-792-2581; Fax: 713-794-4672; E-mail:
kchang@mail.mdanderson.org.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M211849200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PML, promyelocytic
leukemia protein;
APL, acute promyelocytic leukemia;
PML NB, PML
nuclear body;
TNF, tumor necrosis factor
;
CAD, caspase-activated
DNase;
MEFs, mouse embryo fibroblasts;
RAR
, retinoic acid receptor
;
IFNs, interferons;
PCD, programmed cell death;
DMEM, Dulbecco's
modified Eagle's medium;
PBS, phosphate-buffered saline;
PIPES, 1,4-piperazinediethanesulfonic acid;
ELISA, enzyme-linked immunosorbent
assay;
TUNEL, terminal dUTP nick-end labeling;
mAb, monoclonal
antibody;
HA, hemagglutinin;
ICAD, inhibitor of CAD;
DFF, DNA
fragmentation factor.
![]() |
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