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Pathwaysto CTL Involves Fas and Mitochondrial p53 Potentiation of Tumor Cell Susceptibility

Mami-Chouaib and Salem ChouaibAbdelali Jalil, Catherine Richon, Isabelle Vergnon, Fathia Jérôme Thiery, Soraya Abouzahr, Guillaume Dorothee,

http://www.jimmunol.org/content/174/2/871doi: 10.4049/jimmunol.174.2.871

2005; 174:871-878; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/174/2/871.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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p53 Potentiation of Tumor Cell Susceptibility to CTL InvolvesFas and Mitochondrial Pathways1

Jerome Thiery, Soraya Abouzahr, Guillaume Dorothee, Abdelali Jalil, Catherine Richon,Isabelle Vergnon, Fathia Mami-Chouaib, and Salem Chouaib2

In this study, we have investigated the mechanisms used by wild-type p53 (wtp53) to potentiate tumor cell susceptibility toCTL-mediated cell death. We report that wtp53 restoration in a human lung carcinoma cell line Institut Gustave Roussy (IGR)-Heu, displaying a mutated p53, resulted in up-regulation of Fas/CD95 receptor expression associated with an increase of tumorcell sensitivity to the autologous CTL clone, Heu127. However, when IGR-Heu cells were transfected with Fas cDNA, no poten-tiation to Heu127-mediated lysis was observed, indicating that induction of CD95 is not sufficient to sensitize target cells to CTLkilling. Importantly, our data indicate that the effect of wtp53 on the Fas-mediated pathway involves a degradation of shortcellular FLICE inhibitory protein resulting in subsequent caspase 8 activation. Furthermore, we demonstrate that wtp53 resto-ration also resulted in CTL-induced Bid translocation into mitochondria and a subsequent mitochondrial membrane permeabi-lization leading to cytochrome c release. These results indicate that tumor cell killing by autologous CTL can be enhanced bytargeting degranulation-independent mechanisms via restoration of wtp53, a key determinant of apoptotic machineryregulation. The Journal of Immunology, 2005, 174: 871–878.

T he CD8� CTLs have been described to play a crucial rolein host defense against malignancies in both mouse andhuman (1). However, it is also becoming clear that neo-

plastic cells can escape or resist to the immune response at mul-tiple levels. Accumulating evidence suggests that in vivo tumorcell growth is not only influenced by CTL tumor cell recognition,but also by the susceptibility of the tumor cells to host-mediatedimmune response, including killer cells. It is established that an-titumor CTL response is regulated at several effector-target inter-action levels involving both intracellular and extracellular stimuli(2). In this regard, we have previously shown that p53 is a keydeterminant able to improve the effectiveness of CTL responseinvolving at least in part induction of CD95 expression in tumorcells (3).

It is well established that the tumor suppressor p53, activated inresponse to DNA damage, induces cell cycle arrest and apoptosisthrough transcriptional activation of its target genes, hence playinga central role in tumor suppression. P53 has a major function intransducing stress to the apoptotic machinery of the cell, consistentwith the importance of p53 status as a determinant of cellular re-sponse to DNA damaging drugs (4–7). Inactivation of p53 hasbeen identified in many types of tumors particularly in non-smallcell lung carcinoma, one of the most common cancers in which

p53 mutation has been frequently identified. The resultant mutantproteins are frequently functionally compromised for apoptosis in-duction. Such a high mutation frequency suggests a strong selec-tion for loss of wild-type p53 (wtp53)3 function during tumoro-genesis. The ability of p53 to regulate the cell cycle has beenreported to contribute to drug resistance and to apoptosis inducedby many anticancer agents (7). However, it is not known how p53contributes to the immune responses that lead to tumor cell lysis bykiller cells. The direct role of p53 in the control of tumor suscep-tibility to CTL-mediated lysis is still poorly documented and sofar, the link between p53 status and tumor susceptibility to CTLmediated killing remains to be elucidated.

In T cell-mediated cytotoxicity process, two major pathways areengaged following TCR recognition of Ag/MHC complexes ex-pressed on target cells. The first one is a secretory pathway in-volving receptor-triggered exocytosis of preformed secretory gran-ules containing granzymes and perforin (8, 9). The second is basedon receptor-induced surface expression of death receptor ligandson effector cells, which cross-links the corresponding receptors(Fas, TRAIL receptors, TNFR I-p55) on target cells (10). Fas(CD95) pathway plays a major role in CTL-mediated cell death.Upon engagement of Fas by its ligand FasL, Fas-associated deathdomain engages caspase 8, which in turn, autoactivates itself andcleaves downstream substrates directly via activation of thecaspase cascade (mitochondria independent) or indirectly via mi-tochondria-dependent mechanisms. These two mechanisms corre-spond to the predominant arms of CD95 signaling, allowing ef-fector caspases 3, 6, and 7 activation and are thought to operatedistinctly in two distinct types of cells. Cells using the formerpathway are known as type I cells, whereas those using the mito-chondrial pathway are known as type II cells (11). The basis of thiscellular preference in type II cells seems to be depending on therelative stoichiometry of cleaved caspase 8, and on the cleavage of

Laboratoire “Cytokines et Immunologie des Tumeurs Humaines,” Unite 487 InstitutNational de la Sante et de la Recherche Medicale, Institut Federatif de Recherche 54,Institut Gustave Roussy, Villejuif, France

Received for publication May 4, 2004. Accepted for publication November 2, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by a grant from the Institut National de la Sante et de laRecherche Medicale (Paris, France), the Association pour la Recherche contre leCancer Grant 4744, Ligue Francaise Contre le cancer, Fondation de France (ComiteTumeurs Solides). J.T. was supported by a fellowship from the Ligue Nationale Fran-caise de Recherche Contre Le Cancer.2 Address correspondence and reprint requests to Dr. Salem Chouaib, Laboratory“Cytokines et Immunologie des Tumeurs Humaines,” Unite 487 Institut National dela Sante et de la Recherche Medicale, Institut Gustave Roussy, 39 Rue Camille Des-moulins, F-94805 Villejuif Cedex, France. E-mail address: chouaib@igr.fr

3 Abbreviations used in this paper: wtp53, wild-type p53; cFLIP, cellular FLIP; DISC,death-inducing signaling complex; IAP, inhibitor of apoptosis gene.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

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Bid protein into truncated Bid, which links caspase 8 to mitochon-dria pathway (12). Because type II cells depend on the mitochon-drial branch, apoptosis can be blocked by ectopic overexpressionof Bcl-xL or Bcl-2 (13).

Several endogenous inhibitors of Fas-mediated apoptosis havebeen identified, including the cellular FLIP (cFLIP). cFLIP is over-expressed in various tumors and has been shown to be implicatedin tumor resistance to T cell-mediated apoptosis in vivo. Thiscaspase homologue was identified as a potent inhibitor of apopto-sis (IAP) induced by all death receptors (Fas, TRAIL receptors,TNFR I-p55) and was described to allow tumor escape from theimmune response in mouse tumor models (14). Multiple splicevariants of cFLIP have been reported, but so far only two, desig-nated cFLIP short and cFLIP long, could be detected at the proteinlevel (11). The mechanism of cell death inhibition by cFLIP is notcompletely elucidated, but it was suggested that cFLIP, as a com-petitive inhibitor, precludes recruitment of procaspase 8 to thedeath-inducing signaling complex (DISC) and thereby prevents itsactivation (15).

The aim of the present study was to determine the molecularbasis of p53-induced sensitization of target cells to CTL-mediatedkilling. We first demonstrated that restoration of Fas on target cellswas not sufficient to induce potentiation of tumor cells to CTLkilling. Indeed, we have shown that restoration of wtp53 functionresulted in Fas expression induction accompanied with cFLIPshort inhibition and caspase 8 activation. Conversely, we showed,for the first time, that functional interaction between CTL and tu-mor target cells expressing wtp53 resulted in the activation of mi-tochondria leading to Bid translocation, cytochrome c release, andDNA fragmentation. Together, our results emphasize that in addi-tion to its role in controlling irradiation and drug responses, p53also plays a key role in the control of tumor susceptibility to CTL-mediated killing and that its mutation may be associated at least inpart with tumor escape from the immune response. Our studiespoint to the importance of wtp53 function in sensitizing tumorcells, displaying mutated p53, to CTL response and suggest thatthe status of the p53 gene may be an important determinant in theefficacy of antitumor killer cells and may take place in immuno-therapeutic strategies design.

Materials and MethodsAntibodies

Monoclonal Abs directed against Fas (UB2 and ZB4, mouse IgG1; 7C11,mouse IgM) and against caspase 8 (5F7, mouse IgG2b) were purchasedfrom Immunotech. Anti-caspase 3/CPP32 (mouse IgG2a) was purchasedfrom BD Biosciences. Anti-p21WAF1/CIP (Ab1) and anti-p53 (Ab2) werepurchased from Oncogene Research Products. Anti-survivin (C19), antiBcl-xL (S18), anti-Bid (FL195), anti-Bax (N20), anti-actin (C11), anti-cIAP2 (H85), and anti-Bcl2 (100) mAbs were purchased from Santa CruzBiotechnology. Anti-cFLIP (NF6) was purchased from Apotech. Anti-cy-tochrome c was purchased from BD Pharmingen and anti-Hsp70 fromStressGen Biotechnologies.

Tumor cell line and CTL clone

The Institut Gustave Roussy (IGR)-Heu tumor cell line was establishedfrom a patient suffering from a large-cell carcinoma of the lung (16) andwas cultured at 37°C (5% CO2) in DMEM:F12 1:1 with GlutaMax (In-vitrogen Life Technologies) supplemented with 10% FBS (Invitrogen LifeTechnologies) and 5% Ultroser G (BioSera). Heu127 CTL clone was iso-lated from autologous tumor infiltrating lymphocytes as previously de-scribed (17) and was cultured at 37°C (5% CO2) in complete medium(RPMI 1640 with GlutaMax) (Invitrogen Life Technologies) supplementedwith 1% sodium pyruvate (Invitrogen Life Technologies), 10% human sera(Institut Jacques Boy) and rIL-2 in the presence of the autologous tumorcell line and irradiated allogeneic lymphoblastoid cell line.

Construction of recombinant adenovirus vector expressingwtp53 (Adwtp53) and adenovirus infection of cells

Adwtp53 was produced by in vitro homologous recombination in 293 cellsas described (18). Efficiency of Adwtp53 to transfer and direct expressionof wtp53 was analyzed on p53 negative SAOS cells using specific mAb36 h after infection with Adwtp53.

For infection of tumor cell lines, the medium of subconfluent cellsgrown in 10 cm2 culture dishes was removed and 50 PFU per cell of eitherAdwtp53 or empty adenovirus, used as control, was added on 2 ml ofmedium. After 2 h incubation at 37°C, 3 ml of complete medium wereadded to the cells. Cells were cultured for 24 h. The expression of func-tional p53 in Adwtp53 infected cells was then analyzed using Western blotanalysis of p21WAF1/CIP expression and cell cycle progression.

Flow cytometry analysis

Cells (3 � 105) were incubated with UB2 anti-Fas or isotypic control Absfor 60 min at 4°C, followed by FITC-conjugated goat anti-mouse Ab. Theywere analyzed on a FACSCalibur flow cytometer, and data were processedusing CellQuest software (BD Biosciences).

Determination of cell viability with SubG1 assay

Tumor cell (5 � 103) were seeded in flat-bottom 96-well plates and theninfected with Adwtp53 or empty virus for control. After 24 h, 7C11 anti-Fas mAb were added for 6 h. Apoptotic cell death induced by anti-Fas mAbwas analyzed by measuring proportion of subG1 cells. Briefly, cells wereharvested 24 h after infection, washed and fixed in 70% ethanol. Beforeflow cytometry analysis, cells were washed with PBS and stained with 1 mlof propidium iodide (20 �g/ml) containing 100 �g/ml RNase and 20 mMEDTA. DNA content was determined by a FACSCalibur flow cytometer(BD Biosciences) and the proportion of cells in a particular phase of cellcycle was determined by CellQuest software.

Western blot analysis

Total cellular extracts were prepared by lysing cell in ice-cold buffer (50mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 10 �g/mlaprotinin, and 10 �g/ml leupeptin). Equivalent protein extracts (50 �g)were denatured by boiling in SDS and 2-ME, separated by SDS-PAGE andtransferred onto Hybond membranes (Amersham Biosciences). Ponceaured staining of the membranes assessed the efficiency of the electrotransfer.Blots were blocked overnight with TBS containing 5% nonfat dry milk andprobed with appropriated Ab for 1 h. After washing, blots were incubatedwith appropriate secondary Ab-HRP conjugated. The complexes were de-tected using ECL detection kit (Amersham Biosciences).

Cytotoxicity assay

The cytotoxic activity of clone Heu127 was measured by a 6 h lactatedehydrogenase release assay using a Cytotox 96 Non-Radioactive Cyto-toxicity Assay kit (Promega) on 3 � 103 target cells/well. Functional ef-fects of ZB4 anti-Fas neutralizing mAb was tested by preincubating targetcells for 2 h at 37°C before the assay. Experiments were performed inquadruplicate and percentage lysis was determined by 490 nm OD mea-surement as described in the manufacturer notice.

Detection of caspases 3, 7, and 8 activation

Caspase activity was measured by flow cytometry using a CaspaTagCaspase 8 (LETD) Activity kit (Intergen) and a CaspaTag Caspase 3 andCaspase 7 (DEVD) Activity kit (Chemicon International). Briefly, cellswere infected by Adwtp53 or empty virus during 24 h and then treated with7C11 anti-Fas mAb for 4 h or incubated with Heu127 CTL clone for 30min (E:T ratio 3:1). After treatment, the cells were washed, and 106 tumorcells/ml were incubated with FAM-Peptide-FMK for 1 h at 37°C under 5%CO2. After staining, the cells were washed two times and fixed with formal-dehyde-based fixative solution provided in the kit. Flow cytometry analysiswas done using a FACSCalibur flow cytometer (BD Biosciences).

Confocal scanning immunofluorescence microscopy

Cells cultured on coverslips were infected for 24 h and then coincubatedwith H127 CTL clone (E:T ratio 1:1) for 1 h. Cells were washed with PBS,fixed with paraformaldehyde (4% w/v in PBS) for 1 h and then perme-abilized with SDS (0.1% w/v in PBS) for 10 min. Nonspecific sites wereblocked with FCS 10% in PBS for 20 min before the staining with anti-cytochrome c mAb (mouse IgG1; BD Biosciences) and anti-Bid (FL195)pAb (rabbit; Santa Cruz Biotechnology); all were detected by an AlexaFluor 546 (red) goat anti-mouse or an Alexa Fluor 488 (green) goat

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anti-rabbit Abs (Molecular Probes). Nucleus staining was performed withTO-PRO-3 (Molecular Probes). The coverslips were mounted on glassslide using a drop of Vectashield hard set (Vector Laboratories). The flu-orescence was examined under LSM 510 confocal microscope (Zeiss).Z-projection of slices was realized using LSM Image examiner software(Zeiss). A punctuate cytoplasmic staining pattern indicates mitochondriallocalization of cytochrome c or Bid, when a diffuse staining indicates cy-toplasmic localization.

Detection of mitochondrial transmembrane potential disruption

Mitochondrial transmembrane potential (��m) disruption was measuredusing MitoTracker red (chloromethyl X-rosamine (CMX-Ros); MolecularProbes). Briefly, IGR-Heu cells were infected with 50 PFU of control ad-enovirus (empty virus) or Adwtp53 for 24 h and treated or not with 1 �g/ml7C11 anti-Fas mAb during 6 h or incubated with Heu127 CTL clone for 45min. Changes in ��m were determined by staining the cells with 50 nMCMX-Ros in culture medium for 5 min at 37°C in a humidified atmospherecontaining 5% CO2. Cells were then washed in PBS and analyzed by flowcytometry. The decrease in ��m is seen as a decrease in CMX-Rosfluorescence.

ResultsAdwtp53 infection of Fas expressing target cells increase theirlysis by autologous CTL and anti-Fas Abs

For this study, we have used the human non-small cell lung car-cinoma cell line IGR-Heu, defective for Fas receptor expressionand displaying a mutated p53 (codon 132; type AAG � AAT, i.e.,Lys � Asp), and the autologous CTL clone Heu127, able to lyseIGR-Heu following recognition of a mutated �-actinin 4 peptide ina HLA-A2.1 context (19). As previously described, IGR-Heu cellswere lysed by Heu127 CTL clone through the perforin/granzymespathway (3, 20). Infection of IGR-Heu with Adwtp53 resulted inthe restoration of Fas receptor expression and a subsequent in-crease of tumor cell susceptibility to Fas-mediated apoptosis asshown by the percentage of subG1 cells (Fig. 1A) and also resultedto a potentiation of tumor cell sensitivity to Heu127 CTL-mediatedlysis via Fas pathway as demonstrated by Fas neutralization withZB4 mAb (Fig. 1B). Interestingly, transfection of IGR-Heu with

FIGURE 1. Restoration of wtp53function in IGR-Heu cells increasesFas-mediated apoptosis and potentiatesCTL-mediated killing. A, Analysis ofFas-induced apoptosis by measurementproportion of subG1 IGR-Heu or HF-76infected cells. Twenty-four hours afterinfection of cells with Awtp53 or emptyvirus, 7C11 anti-Fas mAb was added at 1�g/ml during 6 h. The cells were thenstained with propidium iodide and ana-lyzed by flow cytometry. Percentage ofFas-positive cells analyzed by flow cy-tometry is indicated. These data are themean of five independent experiments. B,Analysis of CTL-mediated lysis afterIGR-Heu or HF-76 target cell infectionwith Adwtp53 is shown. Cytotoxic activityof the Heu127 CTL clone was evaluatedusing Cytotox 96 NonRadioactive Assay24 h after infection of target cells withAdwtp53. Heu127 CTLs were added at theindicated E:T ratio for 6 h incubation. Spe-cific implication of the Fas pathway inCTL lysis potentiation was demonstratedafter preincubation of IGR-Heu/Adwtp53cells for 2 h with anti-Fas (ZB4) blockingmAb. These data are representative ofthree independent experiments.

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Fas cDNA and isolation of a Fas positive clone, HF-76, was notassociated with a strong increase in its sensitivity to anti-Fas mAb(Fig. 1A) or to CTL-mediated killing (Fig. 1B) as compared withthe parental cell line IGR-Heu lacking Fas expression. However,following restoration of wtp53, the HF-76 Fas� clone exhibited amuch higher susceptibility to anti-Fas mAb and to the Heu127CTL clone. These results indicate that the sole induction of Fasexpression was not sufficient to lead to an increase of tumor cellsusceptibility to the Heu127 CTL clone and that p53 plays anadditional role, besides its ability to induce Fas expression, relatedto an increase of target sensitivity to CTL-mediated lysis.

The effect of p53 on target cell susceptibility to CTL-mediatedlysis is associated with a selective degradation of cFLIP shortprotein

To investigate the mechanisms used by wtp53 to potentiate tumorcell susceptibility to CTL-mediated killing, we analyzed the ex-pression of genes implicated in the control of the Fas-mediatedpathway including Bcl-2 family member genes and IAP genes. Wefirst asked whether the regulatory effect of p53 on target suscep-tibility to the CTL clone involves down-regulation of antiapoptoticgenes or in contrast an increase of proapoptotic gene expression.Western blot analysis, shown in Fig. 2A, indicates that infection ofIGR-Heu cells with Adwtp53 restores wtp53 transactivation activ-ity, as shown by induction of p21 expression, but has no effect onBcl-2, Bcl-xL, Hsp70, survivin, cIAP2, and Bax expression, ex-cluding an impact of wtp53 on Bcl-2 or IAPs family members. Wethen analyzed the expression of cFLIP, which has been describedas a natural inhibitor of Fas-mediated apoptosis. As shown in Fig.2B, Western blot analysis indicates that both long and short formsof cFLIP were significantly expressed in IGR-Heu cells. Interest-ingly, when these cells were infected with Adwtp53, a dramaticdecrease of cFLIP short was selectively observed, whereas cFLIPlong protein expression was not affected. Similar results were ob-tained with HF-76 (data not shown), suggesting that cFLIP shortdegradation following wtp53 restoration is at least associated withFas pathway potentiation.

Activation of caspase 8 and caspase 3/7 in Adwtp53-infectedIGR-Heu cells after Fas engagement

The recruitment of procaspase 8 to the DISC and its subsequentcleavage and activation is generally thought to initiate the Fasapoptotic cascade and it has been reported that cFLIP precludes therecruitment of procaspase 8 to the DISC and thereby prevents itsactivation. We therefore examined the status of procaspase 8 be-fore and after Fas engagement. Data depicted in Fig. 3A indicatethat procaspases 8 and 3 were equally expressed in parental andAdwtp53-infected cells. Fas engagement with anti-Fas mAb re-sulted in the generation of caspase 8 cleaved intermediate productsof 43 and 41 kDa and in cleavage of caspase 3 only in Adwtp53-infected cells. No such cleavages occurred in parental IGR-Heucells after anti-Fas mAb treatment. In addition, treatment of IGR-Heu cells with the Heu127 CTL clone resulted in the generation ofcleaved caspase 8 but not caspase 3. As opposed, treatment ofIGR-Heu/Adwtp53 cells with the Heu127 CTL clone resulted in anincrease of caspase 8 cleavage as well as cleavage of caspase 3.The specific implication of Fas pathway in the cleavage ofcaspases after CTL treatment was demonstrated by blocking theFas receptor with the anti-Fas (ZB4) mAb. To further emphasizethe activation of caspase cascade following wtp53 infection, cellswere treated with carboxyfluorescein-labeled fluoro-methyl ketonepeptide inhibitors of caspase 8 (FAM-LETD-FMK) and caspases3/7 (FAM-DEVD-FMK). When added to a population of cells, the

dye enters into each cell and covalently binds to the active caspaseheterodimer leading to a green fluorescence signal which directlymeasures the amount of active caspases. Data depicted in Fig. 3Bindicate the presence of active caspase 8 and caspase 3/7 after Fasreceptor engagement by anti-Fas mAb only in Adwtp53 infectedcells. In addition, treatment of IGR-Heu cells with the Heu127CTL clone resulted in a weak activation of caspases 8 and 3 sug-gesting that caspase 8 is partially cleaved but not activated. Asopposed, treatment of IGR-Heu/Adwtp53 cells with the Heu127CTL clone resulted in an increase of caspase 8 and 3 activation,mediated by the Fas pathway as shown by blocking the Fas re-ceptor with the anti-Fas (ZB4) mAb. Thus, these data suggest thatthe restoration of wtp53 function in IGR-Heu cells induces cFLIPshort degradation and therefore potentiates the cleavage and acti-vation of caspase 8 and then caspase 3 activation by Fas receptortriggering.

FIGURE 2. Restoration of wtp53 function in IGR-Heu cells does notaffect Bcl-2 and IAP family members but induces selective cFLIP shortdegradation. A, Western blot analysis of p53-induced or repressed genes inIGR-Heu cells is shown. At 24 h after infection of IGR-Heu cells withAdwtp53 or empty virus, cells were lysated and were subjected to Westernblot analysis using anti-Bax, anti-Bcl-2 anti-Bcl-xL, anti-Hsp70, anti-survivin, and anti-cIAP2 Abs. Anti-p21 mAb was used as wtp53 transac-tivation activity control and anti-actin pAb as protein level control. Thesedata are representative of two independent experiments. B, Analysis ofcFLIP protein expression after infection of IGR-Heu cells with Adwtp53.Modulation of cFLIP short and cFLIP long short by p53 was analyzed byWestern blot 24 h after infection of IGR-Heu cells with 50 PFU per cell ofAdwtp53 or empty virus for control. Actin was used as protein level con-trol. These data are representative of two independent experiments.

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Sensitization of wtp53-infected cells to Fas-mediated apoptosisinvolves mitochondrial membrane permeabilization

It has been shown that death receptors can use the mitochondrialpathway to induce an apoptotic signal in type II cells (21). Evi-dence has also been provided indicating that activation of caspase8 and mitochondria were directly connected by truncated-Bid pro-tein to induce the release of apoptotic factors from the mitochon-dria and the full activation of the effector caspase 3. To determinethe possible involvement of mitochondria in the sensitization ofAdwtp53-infected cells to Fas-mediated apoptosis, cells were in-fected with Adwtp53, incubated with anti-Fas mAb, or incubatedwith the Heu127 CTL clone and subjected to cytofluorometricanalysis of ��m disruption after staining with CMX-Ros. Datadepicted in Fig. 4A indicate that Adwtp53 infection and anti-FasmAb or Heu127 CTL treatment of IGR-Heu cells induced a ��m

disruption. In contrast, no significant ��m was detected in anti-FasmAb or Heu127 CTL-treated IGR-Heu parental cells. Moreover,specific implication of the Fas pathway in mitochondrial mem-brane disruption after CTL treatment was demonstrated by block-

ing the Fas receptor with the anti-Fas (ZB4) mAb. In an attempt toconfirm the effect of Adwtp53 on the mitochondrial pathway afterFas engagement, we assessed the subcellular localization of Bidand cytochrome c by confocal microscopy. Data shown in Fig. 4Bindicate that Bid was cleaved and translocated from the cytosol tothe mitochondria. These data also indicate that cytochrome c wasreleased from the mitochondria intermembrane space to the cyto-plasm following Fas triggering only in Adwtp53 infected cells. Asa control, parental and empty virus-infected IGR-Heu cells did notinduce Bid and cytochrome c relocalization. Thus, it appears thatintroduction of wtp53 into IGR-Heu cells restores the mitochon-drial pathway activation following Fas receptor triggering.

Selective Bid translocation and cytochrome c release in tumorcells displaying wtp53 during CTL-target interaction

To determine whether the autologous CTL clone is able to activatethe mitochondrial pathway, IGR-Heu cells, infected or not withadwtp53, were incubated with Heu127 during 1 h and subjected toan immunostaining for Bid, cytochrome c, and the nucleus. Data

FIGURE 3. Effect of Fas agonist mAband Heu127 CTL treatment on procaspase8 cleavage in IGR-Heu cells. A, At 24 hafter infection of IGR-Heu cells with Ad-wtp53 or empty virus, target cells weretreated with the 7C11 anti-Fas mAb for4 h or incubated with the Heu127 CTLclone for 30 min. Total cell lysate (50 �g)were subjected to Western blot analysisusing anti-caspase 8, anti-caspase 3, or anti-actin mAbs. Cleavage products observedin 7C11 mAb treated or Heu127 incubatedcells correspond to bands of 43 and 41kDa. Specific implication of the Fas path-way in the cleavage of caspases after CTLtreatment was demonstrated after preincu-bation of IGR-Heu/Adwtp53 cells for 2 hwith the anti-Fas (ZB4) blocking mAb.Jurkat cells treated with the anti-Fas mAbfor 2 h were used as procaspase 8 and pro-caspase 3 cleavage positive control. Thesedata are representative of two independentexperiments. B, Analysis of caspase 8 andcaspases 3/7 activation in anti-Fas-treatedcells is shown. IGR-Heu cells were in-fected with control adenovirus (empty vi-rus) or Adwtp53 for 24 h and then treatedwith the 7C11 anti-Fas mAb for 4 h orincubated with the Heu127 CTL clone for30 min. Specific implication of the Faspathway in the cleavage of caspases afterCTL treatment was demonstrated afterpreincubation of IGR-Heu/Adwtp53 cellsfor 2 h with the anti-Fas (ZB4) blockingmAb. Detection of caspase 8 and caspases3/7 activity by flow cytometry was per-formed with the CaspaTag assay kit. Re-sults are expressed as percentage of cellsdisplaying activated caspases. These dataare representative of two independentexperiments.

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depicted in Fig. 5 show that Heu127 CTLs induce mitochondrialpathway activation only in Adwtp53 infected cells. Indeed, Bidtranslocation from the cytoplasm to the mitochondria, cytochromec release from the mitochondria intermembrane space to the cyto-plasm, as well as nucleus fragmentation were observed in wtp53IGR-Heu expressing cells after CTL recognition. This mitochon-drial pathway activation is strictly dependent of Fas receptor trig-gering by the Fas-positive ligand CTL clone Heu127, as demon-strated by the anti-Fas neutralizing ZB4 mAb. These data suggestthat wtp53 is a key regulator of tumor target cell killing by specificCTLs through a regulation of Fas signaling and mitochondrialactivation.

DiscussionAt present, most immunotherapeutic strategies are essentially fo-cusing on cytolytic effector cells such as specific CTL and NKcells. Accumulating evidence has been provided indicating thattumor cells are able to generate resistance to CTL-mediated cyto-toxic pathways. This resistance may be independent from tumorcell recognition by the immune system and may result from limitedantitumor effects of specific CTL (22). In this regard, genetic vari-ability or temporal changes in tumor phenotype are two parametersof critical importance that may need to be evaluated to improvetumor sensitivity to cytotoxic cells. During malignant transforma-

tion of human tumors, p53 mutations are selected for loss of theirapoptotic and tumor suppressor ability, which coincides with lossof their transactivation function (23). Although a number of ap-optosis-related genes that are transcriptionally regulated by p53have been identified, the transcription-independent downstreampathway of p53 remains largely unclear. The main goal of thisstudy was therefore to better examine the influence of p53 on theregulation of tumor cell susceptibility to killer cells and to dissectthe molecular mechanisms by which p53 effectively sensitizes tu-mor cells to CTL-mediated lysis. For this purpose, we have used amodel system including a CTL clone and its autologous tumortarget that express a mutated p53. This cell line does not expressdeath receptors (Fas, TRAIL receptors, TNFR I-p55) and was sen-sitive to CTL killing through the exocytosis pathway involvingdegranulation of perforin and granzyme B.

Multiple molecular mechanisms have been described to accountfor the loss of Fas function in cancer cells, including down-regu-lation of transmembrane Fas following promotor methylation,transcriptional repression, histone acetylation, or alternativemRNA splicing to produce a soluble Fas protein lacking trans-membrane anchor (24). Fas is a frequent target for inactivationduring oncogenesis, and it has been hypothesized that Fas-inducedapoptosis plays a crucial role in the biology and treatment of ma-lignant diseases (25). In the present study, we have demonstrated

FIGURE 4. Activation of the mitochondrial path-way by Fas triggering and the Heu127 CTL cloneafter infection with Adwtp53 of IGR-Heu cells. A,Analysis of mitochondrial transmembrane potentialdisruption using the MitoTracker red (CMX-Ros)mitochondrion selective probe is shown. IGR-Heucells were infected with 50 PFU of control adeno-virus (empty virus) or Adwtp53 for 24 h and treatedor not with 1 �g/ml 7C11 anti-Fas mAb for 6 h orincubated with the Heu127 CTL clone for 45 min.Specific implication of the Fas pathway in the cleav-age of caspases after CTL treatment was demon-strated after preincubation of IGR-Heu/Adwtp53cells for 2 h with the anti-Fas (ZB4) blocking mAb.Changes in ��m were determined by staining thecells with 50 nM CMX-Ros. The decrease in ��m isseen as a decrease in CMX-Ros fluorescence. Thesedata are representative of three independent experi-ments. B, Fas-induced Bid translocation and cyto-chrome c release in Adwtp53-infected cells isshown. IGR-Heu cells were infected with 50 PFU ofcontrol adenovirus (empty virus) or Adwtp53 for24 h and treated or not with 1 �g/ml 7C11 anti-FasmAb for 6 h. Immunostaining was performed withanti-Bid (green), anti-cytochrome c Abs (red), andTO-PRO-3 for the nucleus (blue). Note the translo-cation of Bid from the cytoplasm to the mitochon-dria and the release of cytochrome c from mitochon-dria to cytoplasm in Adwtp53-infected and anti-Fas-treated cells. One representative experiment of fouris shown. White arrows indicate cytochrome c re-lease. The confocal scanning fluorescence micro-graphs are representative for the majority (�70%) ofthe cells analyzed. These data are representative ofthe four independent experiments.

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that besides inducing Fas receptor expression on tumor cells, p53acts at multiple steps to sensitize target cells to CTL-mediated lysisby modulating the transcription of a number of cellular targetgenes. Indeed, our data indicate that the induction of Fas receptorexpression on target cells is not sufficient to potentiate CTL killingbecause transfection of Fas cDNA resulting in a strong expressionof CD95 by target cells displaying mutated p53 did not improveCTL-mediated cytotoxicity. This suggests that in addition to a p53-dependent regulation of Fas expression, p53 is able to sensitizetumors cells to CTL-mediated lysis by interfering with additionalevents involved in the regulation of the Fas apoptosis pathway. Inlight of our findings and given the fact that the level of intracellularcFLIP may regulate the sensitivity of tumor cells to apoptosis trig-gered by a variety of stimuli, such as Fas ligand, we investigatedwhether wtp53 modulates cFLIP expression in target cells leadingto subsequent increase in their susceptibility to CTL. Our datashow that both cFLIP long and cFLIP short forms were expressedin parental cells. Interestingly, a marked decrease in cFLIP shortprotein was observed in Adwtp53 infected cells, raising the pos-sibility that the down-regulation of cFLIP short may render targetcells more sensitive to CTL killing. Furthermore, no cFLIP longprotein degradation was observed after Adwtp53 infection, sug-gesting that the wtp53 effect is independent of its transcriptionalinhibition activity. These observations are concordant with a pre-vious report of Fukazawa et al. (26) indicating that an accelerateddegradation of cFLIP long protein occurred through the ubiquitin-proteasome pathway in p53-mediated apoptosis of human cancercells. To examine the functional significance of cFLIP short down-

regulation for the sensitization of wtp53 infected cells to CTL-mediated killing, we analyzed whether such an event was accom-panied by the activation of proapoptotic mediators such as caspase8 believed to play a central role in propagating death receptor-mediated signals. These studies demonstrated a cleavage of theproximal caspase 8 in the DISC of cells displaying wtp53. Fur-thermore, the activation of caspase 8 was found to deliver a signalto mitochondria following cleavage of Bcl-2 family member pro-tein Bid, which translocates to the mitochondria and triggers cy-tochrome c release. The present study implies that DNA bindingdomain of p53 is a dual-function domain, mediating both the trans-activation of Fas gene and the mitochondrial proapoptotic path-way. This also emphasizes the extranuclear death function of p53and shows that this function is critically involved in mediating theproapoptotic action of p53 at the mitochondria level.

Thus, Fas receptor and mitochondrial pathways may be inter-connected leading to the amplification of CTL-mediated target celldeath. These studies demonstrate for the first time a significant roleof mitochondria in the control of target cell death by CTL andclearly show that p53 acts at multiple levels to potentiate the sus-ceptibility of tumor cell to CTL killing. Our study, demonstratingthe implication of mitochondria in CTL-mediated target cell death,provides a mechanistic basis of transcription-independent apopto-sis by p53 and opens a new avenue toward a comprehensive un-derstanding of mutated p53-expressing tumor escape from T cell-mediated cytotoxicity. We propose that p53 plays a linker roleconnecting death receptors to mitochondria leading to amplifica-tion of the apoptotic machinery in tumor cells. Our data support a

FIGURE 5. CTL recognition of IGR-Heu target cellsinduced activation of the mitochondrial pathway onlywith wtp53 infection. CTL-induced translocation of Bidand cytochrome c in Adwtp53-infected cells is shown.IGR-Heu cells were infected with 50 PFU of controladenovirus (empty virus) or Adwtp53 for 24 h and in-cubated with the Heu127 CTL clone during 1 h. Immu-nostaining was performed with anti-Bid pAb (green),anti-cytochrome c mAb (red), and TO-PRO-3 for thenucleus (blue). Note the translocation of Bid from cy-toplasm to mitochondria, the release of cytochrome cfrom mitochondria to cytoplasm, and the fragmentationof the nucleus in Adwtp53-infected cells in contact withCTL. Black arrows indicate Heu127 CTL. One repre-sentative experiment of three is shown. The confocalscanning fluorescence micrographs are representativefor the majority of CTL tumor cell conjugate analyzed.These data are representative of three independentexperiments.

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model in which p53 plays a role in the cross-talk between theapoptosis pathway involving ligation of the Fas receptor and themitochondrial pathway involving Bid translocation and cyto-chrome c release. Although resistance of tumor targets to killercells is likely to be regulated by multiple factors (27), the data wepresent suggest that p53 is an important determinant of target sen-sitivity to CTL-mediated lysis. In this regard, manipulation of p53status may improve effectiveness of CTL to tumor immunotherapyand the clinical use of compounds able to restore p53 functioncould be a novel strategy for optimizing killer cell-based immu-notherapy of cancer.

AcknowledgmentsWe thank Dr. H. Haddada for providing adenovirus recombinant for wtp53and Y. Lecluse for FACS analysis. We thank G. Kroemer, N. Zamzami,and N. Larochette for helpful discussions and J. Even for critical reading ofthe manuscript.

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