Promyelocytic Leukemia Protein Sensitizes Tumor Necrosis Factor�-Induced Apoptosis by Inhibiting the NF-�B Survival Pathway*

Received for publication, November 20, 2002, and in revised form, January 10, 2003Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M211849200

Wen-Shu Wu‡, Zhi-Xiang Xu‡, Walter N. Hittelman§, Paolo Salomoni¶, Pier Paolo Pandolfi¶,and Kun-Sang Chang‡�

From the Departments of ‡Molecular Pathology and §Clinical Investigation, the University of Texas M. D. AndersonCancer Center, Houston, Texas 77030 and the ¶Department of Human Genetics, Memorial Sloan-Kettering Cancer Center,New York, New York 10021

The promyelocytic leukemia protein (PML) is agrowth/tumor suppressor essential for induction ofapoptosis by diverse apoptotic stimuli. The mechanismby which PML regulates cell death remains unclear. Inthis study we found that ectopic expression of PML po-tentiates cell death by apoptosis in the tumor necrosisfactor � (TNF�)-resistant cell line U2OS and other celllines. Treatment with TNF� significantly sensitizedthese cells to apoptosis in a p53-independent manner.PML/TNF�-induced cell death is associated with DNAfragmentation, activation of caspase-3, -7, and -8, anddegradation of DNA fragmentation factor/inhibitor ofCAD. PML/TNF�-induced cell death could be blocked bythe caspase-8 inhibitors CrmA and c-FLIP but not byBcl-2. These findings indicate that this cell death eventis initiated through the death receptor-dependent apo-ptosis pathway. PML is a transcriptional repressor ofNF-�B by interacting with RelA/p65 and prevents itsbinding to the cognate enhancer through the C termi-nus. Coimmunoprecipitation and double-color immuno-fluorescence staining demonstrated that PML physi-cally interacts with RelA/p65 in vivo and the twoproteins colocalized at the endogenous levels. Overex-pression of NF-�B rescued cell death induced by PML/TNF�. Furthermore, PML�/� mouse embryo fibroblastsare more resistant to TNF�-induced apoptosis. Togetherthis study defines a novel mechanism by which PMLinduces apoptosis through repression of the NF-�B sur-vival pathway.

The promyelocytic leukemia gene (PML)1 was initially iden-tified through its fusion to retinoic acid receptor � (RAR�)involved at the breakpoint of t(15,17) (q22;q12) chromosomal

translocation in acute promyelocytic leukemia (APL) (1). ThePML-RAR� fusion protein created by this translocation inter-feres with the normal function of PML and the RAR/retinoid Xreceptor pathway and plays an important role in the pathogen-esis of APL (2, 3). PML is a nuclear protein localized in discretesubnuclear compartments designated PML nuclear bodies(NBs) or PML oncogenic domains (4). PML is a primary targetgene of interferons (IFNs) and is widely expressed in almost allcell lines tested (5). PML is a tumor/growth suppressor thatregulates cell cycle progression and induces cell death (6–9).The proapoptotic and growth-suppressing functions of PMLwere demonstrated in vivo using cells obtained from PMLknockout mice. This study showed that PML is required forFas- and caspase-dependent DNA damage-induced apoptosis insplenocytes and is essential for the induction of programmedcell death (PCD) by Fas, tumor necrosis factor � (TNF�), cer-amide, INF�, INF�, and INF� (10). PML also induces acaspase-independent cell death when force-expressed in ratembryo fibroblasts (11). How PML exerts its proapoptoticeffects remains unknown.

The small ubiquitin-like protein SUMO-1 modifies PML atthree lysine residues (12). This modification appears to beessential for the integrity and function of the PML NBs. Sev-eral reports (13) documented that modification of PML bySUMO-1 is required for the formation of PML NBs. A PMLmutant lacking the SUMO-1 sites did not form PML NBs.Reintroduction of the wild-type PML but not the SUMO-1mutant PML into the PML�/� mouse embryo fibroblasts(MEFs) led to the reorganization of the PML NBs (14, 15). Thisstudy convincingly demonstrated that SUMO-1 modification ofPML is necessary for the NB formation. Recent studies (16, 17)also showed that SUMO-1 modification of PML is essential forinteraction and regulation of transcriptional repression func-tion of Daxx.

Apoptosis is a genetically controlled suicide process consist-ing of two phases. The first phase is characterized by a com-mitment to cell death; the second phase, an execution phase, ischaracterized by membrane inversion, exposures of phosphati-dylserine residues, blebbing, chromatin condensation, andDNA fragmentation. Two major apoptotic pathways, the mi-tochondria-dependent pathway and the death receptor-mediated pathway, have been well documented (18–20). Theactivation of an apoptotic pathway does not necessarily resultin cell death because nuclear factor NF-�B, which up-regulates antiapoptotic genes that block cell death, is alsofrequently activated. For example, the activation of TNFreceptor results in caspase-8 processing, leading to the induc-tion of cell death, whereas NF-�B activation induced by TNFinhibits cell death (21, 22). Therefore, the sensitivity of cellsto apoptotic signals depends on both the NF-�B-mediated

* This work was supported by Grant CA 55577 from the NationalInstitutes of Health (to K.-S. C.). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

� To whom correspondence should be addressed: Dept. of MolecularPathology, 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.

1 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, inter-ferons; PCD, programmed cell death; DMEM, Dulbecco’s modified Ea-gle’s medium; PBS, phosphate-buffered saline; PIPES, 1,4-pipera-zinediethanesulfonic acid; ELISA, enzyme-linked immunosorbentassay; TUNEL, terminal dUTP nick-end labeling; mAb, monoclonalantibody; HA, hemagglutinin; ICAD, inhibitor of CAD; DFF, DNA frag-mentation factor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 14, Issue of April 4, pp. 12294–12304, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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survival pathway and the proapoptotic pathways. Consistentwith this notion, c-Myc, E1a, and E2F1, which inhibit theNF-�B-mediated signaling pathway, enhanced PCD (23–25).

Our results show that PML induces PCD by a death receptor-mediated apoptotic pathway and sensitizes cells to apoptosisupon treatment with TNF� by inhibiting the NF-�B survivalpathway. PML represses NF-�B function by interfering with itsbinding 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 func-tion as a tumor suppressor and define a novel mechanism bywhich PML induces apoptosis through repression of the NF-�Bsurvival pathway.

EXPERIMENTAL PROCEDURES

Plasmids Construction—The full-length PMLIV cDNA was obtainedfrom Professor Pierre Chambon (Institut de Chimie Biologique, Stras-bourg Cedex, France). The inducible expression plasmids pMEP4/PMLand pMEP4/PML(1–555) were constructed by subcloning the PMLcDNA or PML(1–555) into the NotI/XhoI sites of the pMEP4 vector(Invitrogen). The plasmid pMEP4/PML(97–633) was generated by clon-ing 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 subclon-ing the full-length cDNAs amplified by PCR into the pMEP4 vector. Theexpression plasmids pCDNA3/p65 and pMEP4/p65 were created bysubcloning the full-length cDNA of p65/RelA into the BamHI/XhoI sitesof pCDNA3 and the HindIII/XhoI sites of pMEP4. pCMV/HA-NLS-p65and pCMV/HA-NLS-LacZ were generated by cloning RelA/p65 cDNAand LacZ cDNA, respectively, into the BamHI/XbaI sites ofpCMV2N3T. The pCDNA3.1/PML(97–633) was created by cloning theAvrII/EcoRI DNA fragment into the BamHI and EcoRI sites ofpCDNA3.1HisA. The NF-�B-Luc reporter was obtained from ClontechLaboratories, Inc. (Palo Alto, CA).

Cell Cultures and Reagents—The U2OS, 293T, SiHa, Saos2, PML�/�

MEF, and PML�/� MEF cells were maintained in Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with 10% fetal calf serum. DFFantibody (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 pur-chased from Pharmingen. Caspase-3, cytochrome c, and Bcl-2 antibod-ies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Transfection and Luciferase Reporter Assay—Cells were cultured tosemiconfluence and transfected with the expression plasmids usingFuGENE 6 transfection reagent (Roche Diagnostics). For transfectioninto MEFs, the Effectene reagent (Qiagen, Valencia, CA) was used.Luciferase activity was determined using the luciferase reporter assayaccording to the manufacturer’s protocol (Promega Corp., Madison, WI).

Generation of Stable Cell Lines—U2OS cells were transfected witheach 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 withhygromycin (200 �g/ml) for 10 days to establish the stable clonespMEP4/U2OS, PML/U2OS, CrmA/U2OS, p35/U2OS, c-FLIP/U2OS,and Bcl-2/U2OS, respectively. Inducible expression levels of the respec-tive proteins in each stable cell line were determined by induction withCdSO4 (5 �M) for 20 h followed by immunofluorescent staining andWestern blot analysis.

Cell Death Analysis—Cell death was examined by the cell deathenzyme-linked immunosorbent assay (ELISA) according to the manu-facturer’s protocol (Roche Diagnostics) or by trypan blue exclusionassay. The TUNEL assay was also performed to determine cell deathaccording to the manufacturer’s protocol (Promega Corp.).

Colony Forming Assay—A PML stable cell line in U2OS (103) or thecontrol line (pMEP4 in U2OS) was cultured in 6-well plates in DMEMcontaining 200 �g/ml hygromycin. After 8 h, 5 �M CdSO4 or phosphate-buffered saline (PBS) was added, and the culture was continued for 10days.

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 inice-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 phenylmeth-ylsulfonyl fluoride). Cells were permeabilized for 10 min and assessedby trypan blue exclusion assay and then centrifuged for 5 min (480 � g)at 4 °C. The supernatant contained the cytoplasmic protein. The digi-

tonin-insoluble pellet was resuspended in ice-cold extraction buffer (10mM 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 at5,000 � g, the nuclei were resuspended in nuclear extraction buffer (50mM 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 nuclearmixture 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—Immuno-fluorescence staining was performed as described in our previous report(6). The endogenous colocalization of p65 and PML was determined bydouble-color immunofluorescence staining. Briefly, U2OS and SiHacells were pretreated with leptomycin (5 ng/ml) and then treated withTNF� (10 ng/ml) and induced with interferon � for 24 h. Immunofluo-rescent staining was performed using anti-PML mAb (PG-M3, SantaCruz Biotechnology), anti-p65 polyclonal antibody, and secondary anti-bodies. Images were captured with a Zeiss laser scan confocal micro-scope (LSM 5).

Immunoprecipitation—Immunoprecipitation was performed as de-scribed in our previous report (26). For coimmunoprecipitation of theendogenous PML and p65, Jurkat cells were treated with IFN� (3,000units/ml) for 48 h to induce PML expression and then with TNF� (20ng/ml) to activate p65. A total of 500 �g of nuclear protein was used ineach coimmunoprecipitation assay.

Electrophoretic Mobility Shift Assay—The in vitro translated PML orp65 proteins were synthesized by the TNT-coupled wheat germ trans-lated system (Promega Corp.). Nuclear extracts were prepared fromU2OS stable cell lines or cells treated for 1 h with TNF (20 ng/ml). TheNF-�B probe was prepared by annealing the complementary oligonu-cleotides (5�-AGTTGAGGGGACTTTCCCAGG), and the 3�-recessiveends were labeled with Klenow fragment fill-in reaction. Binding reac-tions contained 5 �g of nuclear extracts, 1 �g of poly(dI�dC), 1 ng ofNF-�B probe (1 � 105 cpm) in 20 �l 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 andthen 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 supershiftassays, 50 ng of unlabeled probe or 1 �g of anti-p65 mAb was added tothe binding reactions, respectively.

RESULTS

PML Sensitizes Cells to Tumor Necrosis Factor �-InducedApoptosis—It is well documented by animal and cell culturemodels that PML is a tumor/growth suppressor. To understandfurther the mechanism of the growth-suppressing function ofPML, we generated stable PML clones in U2OS, a humanosteogenic sarcoma cell line. In these cells, expression of PMLis driven by the metallothionein promoter, inducible by Cd2� orZn2�. U2OS cells transfected with vector alone were used as acontrol. The colony growth of these cells was significantly in-hibited when PML expression was induced by Cd2� but not inthe control cells (Fig. 1, a and b), demonstrating the growth-suppressing property of PML in U2OS. The inducible expres-sion level of PML in this cell line is comparable with that inSiHa cells after induction with IFN� (Fig. 1a).

U2OS is resistant to low dose treatment with TNF� (27), butapoptosis can be induced by TNF� in the presence of the pro-tein synthesis inhibitor cycloheximide (Fig. 1c). This indicatesthat the TNF receptor-mediated apoptotic pathway is intact inthis cell line. We next investigated whether the induction ofPML expression by Cd2� in PML/U2OS stable clones resultedin cell death. Our results showed that the expression of PMLwas insufficient to induce cell death in a significant number ofcells within 24 h. We next evaluated whether the death recep-tor-mediated apoptotic pathway is utilized in PML-induced celldeath. The results demonstrated that a combination of PMLand TNF� resulted in massive apoptosis (Fig. 1d). Similarresults were observed when ectopic expression of PML wasachieved by infection with recombinant PML adenovirus, Ad-PML (data not shown).

Our results demonstrated a dramatic synergistic effect be-tween PML and TNF� in the induction of apoptosis in U2OS

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cells. To examine whether PML/TNF�-induced apoptosis isassociated with DNA fragmentation, U2OS cells were infectedwith Ad-PML or the control adenovirus Ad-C for 8 h and thentreated with TNF� for an additional 16 h. Cell death wasexamined by TUNEL assay. As expected, PML expressionalone was insufficient to induce cell death in 24 h; a combina-tion of PML and TNF�, however, induced a massive DNAfragmentation (Fig. 2a), suggesting that caspase-activatedDNase (CAD) was activated during PML/TNF�-induced celldeath. We next sought to determine whether similar effectscould be achieved in other cell lines. We found that PMLexpression dramatically sensitized TNF�-induced apoptosis inSaos2, HT1080, and 293T cell lines (data not shown).

Bcl-2, Bcl-xL, and Bax are important antiapoptotic or pro-apoptotic proteins that control the mitochondria-dependentapoptotic pathway (28–32). We evaluated whether PML regu-lates the expression of these proteins in U2OS cells. Our re-sults showed that ectopic expression of PML had no effect onthese proteins (data not shown). This result is in agreementwith a previous report (10) that deletion of the PML gene didnot affect the expression of the Bcl2 family of proteins.

Activation of Initiator/Effector Caspases and CAD Is Associ-ated with PML/TNF�-induced Apoptosis—The results of ourstudy suggest that the death receptor signaling pathway isinvolved in PML/TNF�-induced cell death. To confirm thisnotion, we evaluated whether PML/TNF�-induced cell deathinvolves 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, TNFreceptors recruit pro-caspase-8 via the adaptor protein FADDand subsequently cleave effector caspases such as caspase-3,

-6, and -7. This activates CAD by degrading the inhibitor ofCAD (ICAD). Consequently, DNA fragmentation and apoptosisoccur (33). These active executioners also cleave other cellularsubstrates that are essential for cell survival and responsiblefor the morphologic and biochemical features of apoptosis.

To determine whether CAD is activated in PML-induced andPML/TNF�-induced cell death, we infected the U2OS cells withAd-PML or Ad-C for 8 h. TNF� was then added for an addi-tional 16 h. CAD activation was then detected by examiningthe degradation of DFF/ICAD. Western blot analysis demon-strated that DFF/ICAD was degraded in the PML/TNF�-treated cells in 24 h (Fig. 2b). Initiator caspase-8 and effectorscaspase-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 deathreceptor apoptotic pathway and is capable of processing almostall other caspases (33). Our finding supports the hypothesisthat PML/TNF�-induced cell death involves the death recep-tor-mediated apoptotic pathway.

PML Induces Cell Death through the Death Receptor-medi-ated Pathway—The binding of TNF� with death receptors ac-tivates caspase-8 and processes effector caspases, includingcaspase-3, -6, and -7. Bcl-2 cannot rescue these cells fromTNF�-induced apoptosis in most cell lines. Active caspase-8can also process the proapoptotic Bcl-2 family member Bid (34)that contains only BH3. Truncated Bid translocates to mito-chondria and induces the loss of mitochondria membrane po-tential (��m) and releases cytochrome c from the mitochondrialintermembrane space to the cytosol. The released cytochrome cbinds to Apaf1 and then recruits and processes pro-caspase-9

FIG. 1. PML functions as a proapoptotic protein. a, inducible expression of PML in the PML/U2OS cell line by various concentrations ofCdSO4 for 16 h. Total proteins were isolated from cells treated with the indicated concentration of CdSO4, and Western blot analysis was performedusing the PML polyclonal antibody. Western blotting was also performed using total proteins isolated from SiHa and U2OS cells treated oruntreated with 1000 units/ml interferon-� (INF-�). b, inducible expression of PML repressed colony formation of U2OS cells. PML/U2OS stablecells (5 � 103) and the negative control (pMEP4/U2OS) were spread onto 6-well plates. After incubation for 6 days in the presence of 5 �M CdSO4or 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 �g/ml cycloheximide (CHX) for 2 h. TNF� (10 ng/ml) was then added, and the cells were incubated for an additional16 h. Percent cell death was measured by trypan blue exclusion assay. d, PML sensitizes TNF�-induced apoptosis in U2OS cells. PML/U2OS orpMEP4/U2OS was induced for 8 h with 5 �M CdSO4, and the cells were then treated or untreated with TNF� (10 ng/ml) for 24 h. Cell death wasquantified by cell-death detection ELISA assay (Roche Diagnostics).

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and initiates the downstream caspase cascade (35). Therefore,the caspase-8-mediated pathway can use mitochondria to acti-vate the executioner apoptosis caspase cascade (36). In con-trast, the apoptosis initiated by the mitochondria-dependentapoptosis pathway can be inhibited by Bcl-2/Bcl-XL, whichblocks cytochrome c release (30).

Death receptor-mediated apoptosis is a very complex processand may involve the mitochondria-dependent pathway in mostcells. Recently, several viral and cellular inhibitors of apoptosishave been found that block or halt apoptotic signaling at de-fined points of the apoptotic pathways. Some examples are asfollows: CrmA (the product of cowpox virus cytokine responsemodifier A) (37), which is a powerful specific inhibitor ofcaspase-8; p35 (the 35-kDa protein of baculovirus AcMNPV)(38), which has a broad specificity but is a less powerful inhib-itor of caspases; c-FLIP (the cellular FLICE-inhibitory protein)(39), a specific inhibitor of caspase-8; and Bcl-2, a cellularinhibitor of the mitochondria-dependent apoptotic pathwaythat acts by preventing cytochrome c release. To determine theapoptotic pathways initiated by PML/TNF�, we assessed theability of these apoptosis inhibitors to block PML/TNF�-in-duced apoptosis.

Stable clones of U2OS cells that conditionally expressedCrmA (CrmA/U2OS), p35 (p35/U2OS), c-FLIP (c-FLIP/U2OS),and Bcl-2 (Bcl-2/U2OS) were established for this study. CrmAand p35 substantially suppressed PML/TNF�-induced celldeath within 24 h (Fig. 3a). CrmA exerted a much strongerinhibitory effect than did p35. Cellular FLICE-inhibitory pro-tein c-FLIP, which blocks TNF receptor-mediated apoptosis,also inhibits PML/TNF�-induced cell death (Fig. 3b). In a sim-ilar experiment, Bcl-2 did not inhibit PML/TNF�-induced celldeath, although it retained its ability to block cytochrome crelease from the mitochondria (Fig. 3c). It is interesting to notethat Bcl-2 did not affect pro-caspase-8 processing. This findingis in agreement with the previous reports (40) that showed thatBcl-2 could not block TNF�-induced apoptosis in some types ofcells. Together, these results strongly support the idea thatPML/TNF�-induced cell death depends upon the death recep-tor-mediated pathway.

PML Sensitizes TNF�-induced Apoptosis by Acting as aFunctional Inhibitor of NF-�B—The TNF� signaling eventsactivate the proapoptotic pathway and the antiapoptotic path-ways through the activation of caspase-8 and NF-�B transac-tivation function, respectively (41). NF-�B consists of two sub-

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 infectedwith 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 wasexamined 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 wereresolved in 12% SDS-PAGE. Western blot analysis was performed using the indicated specific antibodies. The same blots were reprobed withanti-actin or �-tubulin antibody to serve as an internal control. c, activation of caspase-8 protease in the PML/TNF�-treated cells. Cells wereinfected 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 assaywas performed using IETD-p-nitroanilide substrate according to the manufacturer’s protocol (Clontech Laboratories, Inc.). Results shownrepresent an average value of two independent experiments.

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units (p65 and p50 or p52) localized in the cytoplasm to form aninactive complex with an inhibitor of NF-�B (I�B) (41). TNF�induces I�B kinase activation that leads to phosphorylationand subsequently degradation of I�B by proteosome. NF-�Bthen enters the nucleus and activates the transcription of var-ious target genes, including those encoding the antiapoptoticproteins (22, 42). Therefore, it is possible that PML sensitizesTNF�-induced cell death by interfering with NF-�B signaling.

To determine whether PML attenuates signaling of NF-�Bwhen cells are treated with TNF�, we cotransfected an NF-�B-dependent luciferase reporter with the PML expression plas-mid. PML dramatically repressed the transactivation of NF-�Binduced by TNF� (Fig. 4a). This finding suggests that PML isa negative regulator of the NF-�B signaling pathway.

We next evaluated whether PML is a transcriptional repres-sor of NF-�B activity. A series of cotransfection experimentswas performed using RelA (p65), PML expression plasmids,and the NF-�B reporter construct. This study demonstratedthat PML significantly repressed the transcriptional activity ofRelA (p65) (Fig. 4b). In addition, PML inhibited RelA-mediatedtranscription in a dose-dependent manner (Fig. 4c). In a similarexperiment, we found that cotransfection of PML up-regulatesc-Myc-mediated transactivation, indicating that the repressioneffect 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 forsuch inhibitory effect is that PML interacts with RelA andinterferes with its binding to the promoter of target genes.Electrophoretic mobility shift assay demonstrated that PMLsignificantly inhibited RelA binding to its consensus enhancersequence when the in vitro translated proteins (Fig. 5a) or thenuclear extracts were used in the assay (Fig. 5b). To determinewhether PML could stabilize I�B or affect the expression ofRelA/p65 during TNF�-triggered signaling, Western blottingwas performed using total protein isolated from PML/U2OSand pMEP4/U2OS induced with TNF�. This study showed thatPML had little effect on the stability of I�B and expression ofRelA/p65 (Fig. 5c).

In Vivo Association of PML and RelA/p65—To investigatewhether PML and RelA/p65 are associated in vivo, we firstperformed coimmunoprecipitation experiments using total cellextracts isolated from cells cotransfected with PML and RelAexpression plasmids. PML was coimmunoprecipitated and as-sociated with RelA in vivo (Fig. 6a). Our study further showedthat the endogenous RelA and PML were coimmunoprecipi-tated from the nuclear extracts isolated from cells induced byinterferon-� and pretreated with TNF� (Fig. 6b). These resultsstrongly suggest that the two proteins are associated in vivo.

Because PML assembles NB by recruiting other factors inthe cells, our results suggest that PML may recruit RelA into

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 �M) and then infected with Ad-PML. After 8 h, infected cells weretreated 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�-inducedcell 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 abefore analysis of cell death. c, Bcl-2 blocked cytochrome c released from the mitochondria but not caspase-8 processing and cell death induced byPML/TNF�. The indicated inducible-stable cell lines were treated as described in a. Cell death was determined by trypan blue exclusion assay. Thecytosolic and total proteins were extracted and resolved in a 10% SDS-PAGE. Western blot analysis of the cytosolic protein was performed withthe antibodies against cytochrome c, caspase-8, and �-tubulin. Western blot analysis of total proteins was performed using the rabbit anti-PMLantibody.

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the PML NB. To evaluate this possibility, we performed im-munofluorescent staining and confocal microscopy of U2OScells cotransfected with the expression plasmids of PML or

PML mutant and RelA. This study demonstrated that RelAwas indeed recruited to the PML NB in vivo in the cotransfec-tion experiment (Fig. 6c). The mutant PML(1–555), however,

FIG. 4. PML is a transcriptional repressor of RelA (p65). a, PML inhibits activation of NF-�B induced by TNF�. U2OS cells werecotransfected 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/�-Galwas included in each transfection, and �-galactosidase activity was determined to normalize transfection efficiencies. b, PML represses thetranscriptional activation mediated by NF-�B. (NF-�B)-TATA-Luc reporter (0.3 �g) was cotransfected with 0.6 �g of pCDNA3 or pCDNA3/PMLand the indicated amounts of pCDNA3/p65 into U2OS cells. Luciferase activity was determined after 24 h as described in a. c, PML inhibitstranscriptional activity of NF-�B in a dose-dependent manner. (NF-�B)-TATA-Luc or control reporter UAS-TATA-Luc was cotransfected withpCMV/p65 (20 ng) and an increasing concentrations of pCDNA3/PML into U2OS cells. The pCMV/�-gal was included in all transfection assays tomonitor transfection efficiency. Luciferase activity in each assay was determined and normalized by �-galactosidase activity after 24 h. d, PMLenhances Myc-mediated transactivation of reporter in a dose-dependent manner. The reporter plasmid pMyc-TA-Luc was cotransfected withpcDNA3/c-myc and various concentrations of pcDNA3/PML into U2OS cells. Luciferase activity was determined after 24 h post-transfection asdescribed in a. The pCMV/�-Gal was included in each transfection assay to normalize transfection efficiency.

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 �l of in vitro translated p65 and an increasing concentration of in vitro translated PMLprotein. b, PML inhibits NF-�B activation in response to TNF� in vivo. The inducible PML or pMEP4 (vector) stable lines in U2OS were pretreated with5 �M 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 byelectrophoretic 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 �M CdSO4for 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.

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cannot relocate RelA into the PML NB, and the negative con-trol LacZ was not recruited into PML NB indicating that RelA/p65 targeting into the PML NB should be specific. These re-sults support the idea that PML and RelA may be associated inthe PML NB in vivo and that the C terminus of PML is requiredfor such association. Since RelA/p65 mainly localizes to thecytoplasm before treatment with TNF� or other stimuli, it isimportant to examine whether PML can also recruit the endog-enous RelA/p65 to the PML NB when RelA/p65 is translocatedinto the nuclei after TNF� treatment. To evaluate this possi-bility, SiHa and U2OS cells were pretreated with leptomycin B,which has been shown to retain RelA/p65 in the nuclei, andthen were treated with TNF� and interferon. Double-colorimmunofluorescent staining and confocal microscopy detectedcolocalization of the endogenous RelA/p65 and PML in the PMLNB (Fig. 6d) in both the SiHa and U2OS cells. These resultsstrongly support the idea that PML and RelA/p65 are function-ally associated in vivo.

The C Terminus of PML Is Indispensable for Inhibition ofNF-�B—Our study demonstrated that PML functionally re-presses RelA/p65 by recruiting it to the PML NB and interfer-ing with binding of NF-�B to its enhancer. We next attemptedto identify which domain of PML is required to repress NF-�Btransactivation. By using several PML mutants in a series ofcotransfection experiments with the NF-�B reporter construct,we found that PML mutants with a deletion of the RING regionwere capable of inhibiting NF-�B transactivation, whereasPML mutants lacking amino acids 555–633 (PML(1–555)) and305–633 (PML(1–305)) were not. This result indicated the Cterminus of PML is essential for PML to fully inhibit NF-�B

(Fig. 7a). Interestingly, a previous report showed that the Cterminus of PML is required for interactions with p53 andrelocation of p53 to the PML NB (62). To determine whetherthe ability of the PML to inhibit NF-�B is required for theproapoptotic function of PML, a stable cell line expressingPML(1–555) was established in U2OS cells. We compared thesensitivity to TNF� treatment of this stable cell line with thatof the wild-type PML. Our results demonstrated that the Cterminus of PML is required for sensitizing TNF�-inducedapoptosis (Fig. 7b). Result presented in Fig. 6c showed thatPML(1–555) failed to recruit p65 to the PML NBs. We nextperformed coimmunoprecipitation assay to investigatewhether PML(1–555) physically interacts with p65. This studyindeed demonstrated that PML(1–555) was unable to coimmu-noprecipitate p65 (Fig. 7c). Taken together, these results dem-onstrated that the C terminus of PML (amino acids 556–633) isresponsible for inhibiting NF-�B transactivation, recruitingNF-�B to the PML NB, and enhancing TNF�-inducedapoptosis.

RelA Overexpression Blocked PML/TNF�-induced Apo-ptosis—If PML sensitizes U2OS cells to TNF�-induced apo-ptosis by inhibiting the NF-�B-mediated survival pathway asour studies suggest, then ectopic expression of RelA/p65 shouldblock PML/TNF�-induced apoptosis. To test this hypothesis, aRelA-inducible stable cell line was established in U2OS cellsand was infected with Ad-PML or Ad-C in the presence orabsence of CdSO4 and then treated with TNF�. Apoptosis wasthen quantified by a DNA fragmentation assay. The result ofthis study demonstrated that overexpression of RelA signifi-cantly reduced PML/TNF�-induced apoptosis (Fig. 8). This

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 withanti-HA monoclonal antibody or with anti-PML polyclonal antibody. The precipitated complex was resolved in an 8% SDS-PAGE and probed withanti-PML antibody or anti-HA mAb, respectively. b, association of endogenous PML and RelA (p65). PML proteins expression were induced withIFN� for 48 h in Jurkat T cells and then treated with TNF� for 45 min. Nuclear proteins were extracted and coimmunoprecipitated (Co-IP) withanti-p65 mAb (left panel) or with anti-PML polyclonal antibody (right panel). The precipitated complex was resolved by 8% SDS-PAGE and probedwith anti-PML antibody or anti-p65 antibody, respectively. WB, Western blot. c, PML recruits RelA/p65 into PML NB. The pCDNA3/PML andpCMV/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 sequentiallyimmunostained with anti-HA mAb and anti-mouse IgG-conjugated with rhodamine and then with anti-PML antibody and anti-rabbit IgGconjugated with fluorescein isothiocyanate. HA-LacZ was transfected and stained as a negative control. d, confocal microscopic analysis of theendogenous colocalization of p65 and PML in Siha and U2OS cells. Cells were pretreated with leptomycin (5 ng/ml) and TNF� (10 ng/ml) and thenwere induced with interferon � for 24 h. Cells were stained with anti-PML mAb and anti-p65 polyclonal antibody followed by fluoresceinisothiocyanate-conjugated or rhodamine-conjugated secondary antibody.

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PML/TNF�-induced cell death was associated with DFF/ICADdegradation. These results demonstrated that PML is a func-tional inhibitor of RelA/p65.

Loss of PML Function Renders Cells Resistant to TNF�-induced Apoptosis—We next sought to examine the effects ofendogenous PML on TNF�-induced signaling events inPML�/� and PML�/� MEFs. A culture medium with lowgrowth and low survival factors was selected to sensitize thewild-type MEFs to apoptosis in response to TNF� treatment.MEFs derived from PML�/� and PML�/� mice were tested fortheir relative sensitivity to TNF� under similar conditions.PML�/� and PML�/� MEFs exhibited similar survival rates,but the PML�/� MEFs were significantly more resistant thanthe PML�/� MEFs to TNF�-induced apoptosis (Fig. 9a). Thisexperiment was repeated, and similar results were observed.This finding implies that a loss of PML function rendered cellsresistant to TNF�-induced apoptosis.

To provide further support of this finding, we investigatedhow PML expression affected sensitivity to TNF�-mediated celldeath. PML�/� MEFs and PML�/� MEFs were infected withAd-PML and the control Ad-C. The results of this study showedthat reintroduction of PML into the PML�/� MEFs restoredsensitivity to TNF�-mediated cell death (Fig. 9b). We nextexamined whether TNF� increases activity of NF-�B transac-tivation in PML-deficient cells in a transient transfection as-say. The results demonstrated a moderate but consistent in-crease in reporter activity in the PML�/� MEFs (Fig. 9c).

DISCUSSION

This study shows that PML sensitizes cells to TNF�-inducedapoptosis in U2OS and several other cell lines through the

death receptor-dependent apoptotic pathway. The C terminusof PML is indispensable for the proapoptotic functions of thePML. The proapoptotic function of PML is p53-independent, asshown by the effect of the PML on the p53-negative Saos-2 cellline. By using the TNF�-resistant U2OS cell line as a model,we showed that PML sensitizes TNF�-induced cell death by

FIG. 8. RelA expression blocked PML/TNF�-induced apopto-sis. The RelA/p65-inducible stable cell line was pretreated with 5 �M

CdSO4 for 16 h to induce the expression of RelA. Cells were theninfected with Ad-C or Ad-PML for 8 h and then treated with TNF� (10ng/ml) for 16 h. Cell death was quantified by the cell death detectionELISA. Total proteins were isolated from the same samples and re-solved in a 10% SDS-PAGE. Western blot analysis was performed withanti-DFF/ICAD antibody and anti-tubulin mAb.

FIG. 7. The C terminus of PML is required for inhibition of NF-�B activity and sensitizing TNF�-induced apoptosis. a, the Cterminus of PML is required for complete inhibition of NF-�B activity. NF-�B reporter plasmid was cotransfected with pCDNA3/p65 and theindicated PML mutants into U2OS cells. Luciferase activity was assayed at 24 h post-transfection. b, the C terminus of PML is required forsensitizing cells to TNF�-induced apoptosis. The inducible stable cell lines expressing wild-type PML, PML(1–555), or vector (pMEP4) werepretreated with 5 �M CdSO4 for 16 h. TNF� (10 ng/ml) was then added, and cells were cultured for an additional 24 h. Cell death was thendetermined 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. Coimmunopre-cipitation (IP) and Western blotting (WB) were performed using the anti-HA monoclonal antibody or the PML polyclonal antibody (pAb).

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activating caspase-8, -7, and -3 and degrading DFF/ICAD. Byusing stable cell lines with inducible expression of several ofthe inhibitors of apoptosis, our study showed that PML/TNF�induced apoptosis through the death receptor-mediated path-way. Overexpression of Bcl2 did not effectively inhibit celldeath induced by PML/TNF�, but p35, CrmA, and c-FLIP did.This observation does not support the involvement of the mi-tochondria pathway. The results of this study support thenotion that PML/TNF�-induced apoptosis is mediated throughthe death receptor apoptotic pathway.

NF-�B activation can be found in many different types ofcancers, serving as a mechanism to prevent cancer cell deathand contributing to tumorigenesis (44). We examined whetherapoptosis could be induced through the death receptor-medi-ated pathway by regulating the NF-�B survival pathway. Ourresults (Figs. 4 and 5) demonstrated that PML represses thetransactivation function of NF-�B by interacting with p65/RelAand preventing it from binding to the NF-�B recognition se-quence. This result is in agreement with our previous report(45) that demonstrated that PML repressed A-20-mediatedtranscription, a target gene of NF-�B, through the NF-�B-binding site. Coimmunoprecipitation of PML and p65/RelA wasfound at the endogenous levels and supports an in vivo associ-ation between the two proteins. This notion was further sup-ported by the finding that PML colocalizes with p65/RelA at theendogenous levels in both U2OS and SiHa cells. Our resultsfurther showing that stable overexpression of RelA inhibitsPML/TNF�-induced apoptosis (Fig. 8). Together, these studiesdemonstrated that PML sensitizes TNF�-induced apoptosis byinhibiting the NF-�B survival pathway. Further support forthis conclusion was obtained using the PML knockout MEFs ina TNF�-induced cell death assay. This study showed that PML-deficient cells are more resistant to TNF�-induced apoptosisthan are normal MEFs and that TNF� sensitivity in these cellscan be restored when PML expression is reintroduced by ade-novirus-mediated gene transfer.

It is clear that c-FLIP specifically inhibits caspase 8, anupstream initiator of the TNF-induced death receptor-medi-ated apoptosis. Recent findings (46) demonstrated that c-FLIP

is a target gene of NF-�B and that restoration of FLIP inNF-�B-null cells efficiently inhibits TNF- and FasL-inducedapoptosis. This finding, together with our results presentedhere, demonstrates that PML sensitizes TNF�-induced apo-ptosis by inhibiting the NF-�B transactivation function. Down-regulation of NF-�B transactivation relieves c-FLIP inhibitionof caspase-8, leading to the activation of this apoptosis initiatorand downstream effector caspases. At the same time, othertarget genes of NF-�B, including several of the inhibitor of

FIG. 9. Loss of PML function ren-dered cells resistant to TNF�-in-duced apoptosis. a, the effects of endog-enous PML on TNF� receptor-mediatedapoptosis signaling pathway. PML�/�

MEF and PML�/� MEF were cultured inDMEM containing 0.5% serum and thentreated with TNF� (50 ng/ml) for 48 h.Cell death was quantified by the celldeath detection ELISA. b, reintroductionof PML into PML�/� MEF restored TNF�-mediated cell death. The MEFs andPML�/� MEF were infected with Ad-PMLand Ad-C in the presence or absence ofTNF�. Cell death was quantified as de-scribed in a. c, promoter activity of NF-�Bin PML�/� MEF and PML�/� MEF. TheNF-�B reporter plasmid (NF-�B)-TATA-Luc was transfected into the PML�/�

MEF and PML�/� MEF. Cells were cul-tured for 18 h and then treated with 10ng/ml of TNF� for 4 h. Total proteins wereisolated, and luciferase activity was de-termined as described in Fig. 4 legend.

FIG. 10. A schematic illustration of PML/TNF�-induced apo-ptosis through inhibition of the NF-�B survival pathway. TNF�-induced activation of upstream initiator caspase-8 further activatesseveral of the downstream caspases including caspase-3, -6, and -7.CADs become activated and resulted in DNA fragmentation and even-tually apoptosis. At the same time, TNF� also induced activation of NIKwhich activates NF-�B by induced phosphorylation and degradation ofI-�B. NF-�B in turns activates many of the downstream target genesincluding anti-apoptotic genes cFLIP, IAPs, Bcl-XL, and A20 and pre-vent cells from undergoing apoptosis. PML sensitizes TNF�-inducedapoptosis by acting as a functional inhibitor of NF-�B.

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apoptotic proteins, e.g. IAPs, will also be down-regulated, re-sulting in further activation of effector caspases. Together,these events weaken the NF-�B survival pathway and triggerapoptotic cell death (see Fig. 10). Our studies that overexpres-sion of Rel A and c-FLIP inhibits PML/TNF� induced apoptosis(Fig. 3b and Fig. 8) support this hypothesis.

NF-�B plays a central role in host defense and inflammatoryresponses, and its activity can be activated rapidly by manyproinflammatory agents, including cytokines and virus (47).NF-�B regulates a wide variety of genes, including many of thegenes encoding cytokines, chemokines, and adhesion molecules(47–49). Many of the apoptosis inhibitory proteins, includingIAPs, A1, A20, Bcl-XL, FasL, TRAFs, and c-FLIP, are alsotargets of NF-�B (48, 51). An important role of NF-�B in anti-apoptosis was first demonstrated by Beg et al. (49), who showedthat p65/RelA-deficient mice died in the embryo from extensiveliver apoptosis at E15. NF-�B activity is normally controlled byI�B, a cytoplasmic protein that forms an inactive complex withNF-�B and inhibits NF-�B activity by preventing it from en-tering the nucleus. Our study shows that PML, a nuclearprotein, also inhibits NF-�B activity. Thus NF-�B-mediatedsignaling could be regulated in both the cytoplasm and thenucleus. In addition, TAFII 105 is a nuclear transcriptionalcoactivator of NF-�B essential for induction of antiapoptoticproteins in response to TNF� (52). A dominant-negative TAFII

105 blocked the interaction between NF-�B and TAFII 105 andseverely reduced cell survival in response to TNF� (53). An-other IFN-inducible protein, p202 (50), was shown to inhibitNF-�B activity in the nucleus and TNF�-induced sensitizedcell death through a mechanism similar to that utilized byPML.

PML is involved in viral DNA replication, and it appears thatthe PML NB-associated proteins are released to viral replica-tion and transcription domains. The two early transcribed ad-enovirus proteins E4-open reading frame 3 and E1B (54, 55)are targeted to the PML NB and trigger its dissociation fromother cellular factors or the release of some important factorsthat are required for viral propagation and the prevention ofapoptosis of host cells. The inhibition of the PML NB dissocia-tion reduces adenovirus replication, supporting the idea thatthe PML NB retains cellular factors that play critical roles inviral replication, regulation of viral transcription, and host-cellsurvival. It is well documented that NF-�B is frequently acti-vated by viral infection and plays a critical role in viral onco-genesis and the regulation of viral gene transcription (56). Ourfinding that PML represses NF-�B (p65) transcriptional activ-ity may help explain why the PML NB is the target of severalvirus proteins. The PML NB has been shown to be the target ofadenovirus, human T-cell leukemia virus type 1 (57), papillo-mavirus (58), hepatitis virus (59), herpes simplex virus (60),and human cytomegalovirus (61).

Other mechanisms of PML-induced apoptosis have also beenreported. A role of PML in p53-dependent apoptosis in thymo-cytes has been reported recently (62) in response to ionizationradiation. This pathway involves activation of the p53 down-stream target genes bax and p21. Another pathway of PML-induced apoptosis was also reported recently. This pathway isinduced in response to Fas in B and T splenocytes in a p53-independent manner through a mechanism involve the in vivoassociation between PML and the proapoptotic protein Daxx (9,63). Daxx was originally identified as a Fas death domainbinding protein (64); it also directly interacts with PML andcolocalizes in the PML NBs. Daxx regulation of Fas-inducedapoptosis required the ability of Daxx to colocalize to the PMLNBs (65). Our study here shows that PML/TNF�-inducedapoptosis is a mechanism independent from the p53 function. It

is not clear at this stage whether this mechanism is in anywayconnected with the Fas/Daxx-mediated apoptotic pathway. Ithas been shown that Daxx acts as a transcriptional repressorby recruiting histone deacetylases. The SUMO-1-modified PMLnegatively regulates transcriptional repression of Daxx by in-teracting and sequestering Daxx to the PML NBs (17, 18).Although the detailed mechanism of Fas/Daxx-induced apo-ptosis remains unclear, it is possible that PML regulates thispathway through a direct interaction with Daxx.

PML and its associated proteins play a critical role in thecontrol of cell growth, although the molecular mechanism is notclear. Recent findings (62, 66) demonstrated that p53 wasrecruited to the PML NB through a direct interaction betweenthe core domain of p53 and the C terminus of PML, resulting inenhanced transactivation of p53 in a promoter-specific mannerand affecting cell survival. This study implies that the PML NBis required but alone is insufficient to enhance p53-inducedapoptosis. Although it lacks the C terminus, PML(1–555) formsa nuclear body (67) but cannot enhance p53-mediated celldeath. Interestingly, the C terminus of PMLIV isoform is alsoessential for the interaction with p65 (Fig. 6c and 7c). Moreimportant, the PML mutant lacking the C terminus could notfully inhibit p65 activity and lost its ability to enhance apo-ptosis in response to TNF�. Various PML isoforms have beenfound that share the same N terminus with variable C terminigenerated by alternative splicing (68). We speculate that thevarious functions of PML may be regulated through alternativesplicing to produce various PML isoforms with different cellu-lar functions. Our previous study (26) demonstrated that onlya specific isoform of PML interacts with histone deacetylasesfor transcriptional repression. There is also evidence that onlysome PML isoforms interact with retinoblastoma protein. It istherefore important in the future to study how PML regulatestranscription, cell growth, and apoptosis through specificisoforms.

Acknowledgments—We are grateful to Mariann Crapanzano for ed-iting and critical reading of the manuscript. The DNA sequencing andthe confocal microscopy facilities were supported by NCI ResearchGrant CA-16672 from the National Institutes of Health.

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PML/TNF�-induced Apoptosis12304

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and Kun-Sang ChangWen-Shu Wu, Zhi-Xiang Xu, Walter N. Hittelman, Paolo Salomoni, Pier Paolo Pandolfi

B Survival PathwayκApoptosis by Inhibiting the NF--InducedαPromyelocytic Leukemia Protein Sensitizes Tumor Necrosis Factor

doi: 10.1074/jbc.M211849200 originally published online January 22, 20032003, 278:12294-12304.J. Biol. Chem. 

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