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• W2020838087 abstract "In this study we investigated the involvement of p53 in cytotoxic T-lymphocyte (CTL)-induced tumor target cell killing mediated by the perforin/granzymes pathway. For this purpose we used a human CTL clone (LT12) that kills its autologous melanoma target cells (T1), harboring a wild type p53. We demonstrated initially that LT12 kills its T1 target in a perforin/granzymes-dependent manner. Confocal microscopy and Western blot analysis indicated that conjugate formed between LT12 and T1 resulted in rapid cytoplasmic accumulation of p53 and its activation in T1 target cells. Cytotoxic assay using recombinant granzyme B (GrB) showed that this serine protease is the predominant factor inducing such accumulation. Furthermore, RNA interference-mediated lowering of the p53 protein in T1 cells or pifithrin-α-induced p53-specific inhibition activity significantly decreased CTL-induced target killing mediated by CTL or recombinant GrB. This emphasizes that p53 is an important determinant in granzyme B-induced apoptosis. Our data show furthermore that when T1 cells were treated with streptolysin-O/granzyme B, specific phosphorylation of p53 at Ser-15 and Ser-37 residues was observed subsequent to the activation of the stress kinases ataxia telangiectasia mutated (ATM) and p38K. Treatment of T1 cells with pifithrin-α resulted in inhibition of p53 phosphorylation at these residues and in a significant decrease in GrB-induced apoptotic T1 cell death. Furthermore, small interference RNAs targeting p53 was also accompanied by an inhibition of streptolysin-O/granzyme B-induced apoptotic T1 cell death. The present study supports p53 induction after CTL-induced stress in target cells. These findings provide new insight into a potential role of p53 as a component involved in the dynamic regulation of the major pathway of CTL-mediated cell death and may have therapeutic implications. In this study we investigated the involvement of p53 in cytotoxic T-lymphocyte (CTL)-induced tumor target cell killing mediated by the perforin/granzymes pathway. For this purpose we used a human CTL clone (LT12) that kills its autologous melanoma target cells (T1), harboring a wild type p53. We demonstrated initially that LT12 kills its T1 target in a perforin/granzymes-dependent manner. Confocal microscopy and Western blot analysis indicated that conjugate formed between LT12 and T1 resulted in rapid cytoplasmic accumulation of p53 and its activation in T1 target cells. Cytotoxic assay using recombinant granzyme B (GrB) showed that this serine protease is the predominant factor inducing such accumulation. Furthermore, RNA interference-mediated lowering of the p53 protein in T1 cells or pifithrin-α-induced p53-specific inhibition activity significantly decreased CTL-induced target killing mediated by CTL or recombinant GrB. This emphasizes that p53 is an important determinant in granzyme B-induced apoptosis. Our data show furthermore that when T1 cells were treated with streptolysin-O/granzyme B, specific phosphorylation of p53 at Ser-15 and Ser-37 residues was observed subsequent to the activation of the stress kinases ataxia telangiectasia mutated (ATM) and p38K. Treatment of T1 cells with pifithrin-α resulted in inhibition of p53 phosphorylation at these residues and in a significant decrease in GrB-induced apoptotic T1 cell death. Furthermore, small interference RNAs targeting p53 was also accompanied by an inhibition of streptolysin-O/granzyme B-induced apoptotic T1 cell death. The present study supports p53 induction after CTL-induced stress in target cells. These findings provide new insight into a potential role of p53 as a component involved in the dynamic regulation of the major pathway of CTL-mediated cell death and may have therapeutic implications. Antigen-specific CD8+ T cells play a crucial role in host defense against malignancies in both mouse and human models (1Smyth M.J. Godfrey D.I. Trapani J.A. Nat. Immunol. 2001; 2: 293-299Crossref PubMed Scopus (634) Google Scholar). In the T cell-mediated cytotoxicity process, two major pathways are involved after T cell receptor recognition of silver-major histocompatibility complex complexes expressed on target cells. The first one is an alternate pathway based on T cell receptor-induced surface expression of death receptor ligands (FasL, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), and TNF) on effector cells, which cross-links the corresponding receptors (Fas/CD95, TRAIL receptors, and TNF-RI/p55, respectively) on target cells (2Ashkenazi A. Nat. Rev. Cancer. 2002; 2: 420-430Crossref PubMed Scopus (1108) Google Scholar). The second, which is undoubtedly the major pathway, is a secretory pathway involving receptor-triggered exocytosis of preformed secretory lysosomes, termed lytic granules (3Lieberman J. Nat. Rev. Immunol. 2003; 3: 361-370Crossref PubMed Scopus (556) Google Scholar, 4Trapani J.A. Smyth M.J. Nat. Rev. Immunol. 2002; 2: 735-747Crossref PubMed Scopus (887) Google Scholar). On the basis of findings in genetically manipulated mice, human genetic disease, and in vitro studies, the granule exocytosis pathway seems to have the dominant role in eliminating virus-infected cells and in tumor immunosurveillance (5Kagi D. Ledermann B. Burki K. Seiler P. Odermatt B. Olsen K.J. Podack E.R. Zinkernagel R.M. Hengartner H. Nature. 1994; 369: 31-37Crossref PubMed Scopus (1529) Google Scholar). The cytotoxic granules contain the pore-forming protein perforin and a family of highly specific serine proteases know as granzymes. In mice and humans, A and B are the most abundant granzymes and have received the most attention, in particular granzyme B. It has been suggested that the latter induces target cell death by cleaving and activating the pro-apoptotic Bcl-2 family member Bid (6Barry M. Heibein J.A. Pinkoski M.J. Lee S.F. Moyer R.W. Green D.R. Bleackley R.C. Mol. Cell. Biol. 2000; 20: 3781-3794Crossref PubMed Scopus (286) Google Scholar). Truncated Bid disrupts the outer mitochondrial membrane to cause release of proapoptotic factor cytochrome c and endonuclease G (7Sutton V.R. Wowk M.E. Cancilla M. Trapani J.A. Immunity. 2003; 18: 319-329Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). It has also been suggested that granzyme B (GrB) 5The abbreviations used are: GrB, granzyme B; wt, wild type; CTL, cytotoxic T-lymphocyte; Ab, antibody; Dioc6(3), 3,3′-dihexyloxacarbocyanine; CMA, concanamycin A; PFT-α, pifithrin-α; ROS, reactive oxygen species; siRNA, small interference RNA; PBS, phosphate-buffered saline; SLO, streptolysin-O; ATM, ataxia telangiectasia mutated. may induce target cell death by activating caspase 3 directly, by cleaving caspase substrates like poly ADP-ribose polymerase or inhibitor of caspase-activated DNase (CAD) to free CAD, and/or by cleaving several non-caspase substrates (8Sharif-Askari E. Alam A. Rheaume E. Beresford P.J. Scotto C. Sharma K. Lee D. DeWolf W.E. Nuttall M.E. Lieberman J. Sekaly R.P. EMBO J. 2001; 20: 3101-3113Crossref PubMed Scopus (115) Google Scholar). GrB also directly disrupts the mitochondrial transmembrane potential in a caspase- and Bid-independent manner (3Lieberman J. Nat. Rev. Immunol. 2003; 3: 361-370Crossref PubMed Scopus (556) Google Scholar, 9Thomas D.A. Scorrano L. Putcha G.V. Korsmeyer S.J. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14985-14990Crossref PubMed Scopus (102) Google Scholar). However, despite these advances, the functional relationship between GrB and the tumor suppressor protein p53 remains unknown. It is well established that an appropriate response to stress stimuli is crucial for preventing cellular transformation as well as for maintaining normal tissue function. The tumor suppressor protein p53 has a central role in protecting cells from a variety of stress stimuli such as DNA damage, nucleotide depletion, oncogene activation, or γ-irradiation (10Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2292) Google Scholar). The p53 protein has a short half-life and is often undetectable in normal cells. It is activated as a transcription factor through numerous post-translational modifications that allow its stabilization and accumulation in the nucleus to regulate target gene expression (11Bode A.M. Dong Z. Nat. Rev. Cancer. 2004; 4: 793-805Crossref PubMed Scopus (1022) Google Scholar). Activated p53 induced transcription from promoters that harbor a p53 consensus binding site of genes involved in the maintenance of genetic stability and cellular homeostasis (12el-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1748) Google Scholar). Many apoptosis-related genes are regulated by p53, such as those encoding death receptors (13Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (305) Google Scholar) and the proapoptotic Bcl-2 proteins p53-up-regulated modulator of apoptosis (Puma) (14Nakano K. Vousden K.H. Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1881) Google Scholar) and Noxa (15Oda E. Ohki R. Murasawa H. Nemoto J. Shibue T. Yamashita T. Tokino T. Taniguchi T. Tanaka N. Science. 2000; 288: 1053-1058Crossref PubMed Scopus (1707) Google Scholar). As an additional mode of apoptotic activity, p53 also accumulates in the cytoplasm, where it directly activates the proapoptotic protein Bax to promote mitochondrial outer-membrane permeabilization (16Chipuk J.E. Kuwana T. Bouchier-Hayes L. Droin N.M. Newmeyer D.D. Schuler M. Green D.R. Science. 2004; 303: 1010-1014Crossref PubMed Scopus (1669) Google Scholar). Moreover, it has been reported that in response to a broad spectrum of apoptotic stimuli, a fraction of wtp53 rapidly translocates to mitochondria in cell lines, in primary cells, and in vivo (17Marchenko N.D. Zaika A. Moll U.M. J. Biol. Chem. 2000; 275: 16202-16212Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 18Sansome C. Zaika A. Marchenko N.D. Moll U.M. FEBS Lett. 2001; 488: 110-115Crossref PubMed Scopus (155) Google Scholar). Endogenous mitochondrial p53 physically interacts with the Bcl-2 family member proteins Bcl-XL and Bcl-2 and antagonizes their anti-apoptotic stabilization of the outer membrane (19Mihara M. Erster S. Zaika A. Petrenko O. Chittenden T. Pancoska P. Moll U.M. Mol. Cell. 2003; 11: 577-590Abstract Full Text Full Text PDF PubMed Scopus (1464) Google Scholar). p53 possesses, therefore, a non-transcriptional function that is independent of its nuclear activity (20Vousden K.H. Science. 2005; 309: 1685-1686Crossref PubMed Scopus (72) Google Scholar). The physiological consequences of p53 activation essentially lead to cell cycle arrest, senescence, DNA repair, or apoptosis; thereby, p53 prevents cells from replicating a genetically compromised genome. Moreover, the ability of p53 to regulate the cell cycle and apoptosis has been reported to contribute to drug sensitivity induced by many anti-cancer agents (21Brown J.M. Wouters B.G. Cancer Res. 1999; 59: 1391-1399PubMed Google Scholar). Nevertheless, the role of this tumor suppressor protein in the control of apoptosis mediated by cytotoxic T-lymphocyte (CTL) is not well documented. In this regard we have previously shown that p53 is a key determinant in anti-tumor CTL response as it regulates induction of Fas receptor expression, cellular FLICE/caspase-8 inhibitory protein (cFLIP) short protein degradation, and CD95-induced activation of mitochondrial pathway in tumor cells (22Thiery J. Dorothee G. Haddada H. Echchakir H. Richon C. Stancou R. Vergnon I. Benard J. Mami-Chouaib F. Chouaib S. J. Immunol. 2003; 170: 5919-5926Crossref PubMed Scopus (28) Google Scholar, 23Thiery J. Abouzahr S. Dorothee G. Jalil A. Richon C. Vergnon I. Mami-Chouaib F. Chouaib S. J. Immunol. 2005; 174: 871-878Crossref PubMed Scopus (24) Google Scholar). The present studies were designed to delineate the relationship between p53 and GrB during tumor-specific lysis. We first demonstrated that CTL-tumor target cell interaction resulted in p53 accumulation and activation. Such activation is mediated by GrB and contributes at least in part to GrB-induced apoptosis. The current study emphasizes that in addition to its role in controlling irradiation and drug responses, p53 also plays a key role in the regulation of CTL-mediated apoptosis of tumor cells. Antibodies and Reagents—Antibodies directed against p53 (DO-1, mouse IgG2a), Mdm2 (SMP14, mouse IgG1), Bid (FL195, rabbit IgG), and actin (C11, goat IgG) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-caspase 3 (8G10) rabbit monoclonal Ab, phospho-p53 antibody (Ser-6, Ser-9, Ser-15, Ser-20, Ser-37, Ser-46, and Ser-392), phospho-p38K (Thr-180/Tyr-182), and phospho-ATM (Ser-1981) polyclonal antibodies were from Cell Signaling Technology (Beverly, MA). Recombinant human GrB was purchased from Alexis Biochemicals (Lausen, Switzerland). Tumor Cell Line and CTL Clone—The T1 tumor cell line was established from the primary lesion of a patient suffering from a melanoma (24Dufour E. Carcelain G. Gaudin C. Flament C. Avril M.F. Faure F. J. Immunol. 1997; 158: 3787-3795PubMed Google Scholar) and was cultured at 37 °C (5% CO2) in RPMI 1640 with GlutaMax™ (Invitrogen) supplemented with 5% of fetal bovine serum (Invitrogen) and 5% Ultroser® G (BioSera, France). The LT12 CTL clone was isolated from autologous tumor infiltrating lymphocytes as described previously (24Dufour E. Carcelain G. Gaudin C. Flament C. Avril M.F. Faure F. J. Immunol. 1997; 158: 3787-3795PubMed Google Scholar) and was maintained at 37 °C (5% CO2) in complete medium (RPMI 1640 with GlutaMax™) (Invitrogen) supplemented with 1% sodium pyruvate (Invitrogen), 5% human serum (Institut Jacques Boy, Reims, France), and recombinant interleukin-2 in the presence of the autologous tumor cell line and irradiated LAZ and allogenic peripheral blood mononuclear cells. Cell Death Analysis—T1 tumor cell sensitivity to LT12 was evaluated after interaction lasting 30 min and 1 h by 3,3′-dihexyloxacarbocyanine iodide (Dioc6(3)) and propidium iodide labeling (Molecular Probes, Eugene, OR). Inhibition of the perforin/granzymes-mediated cytotoxicity was performed using LT12 cells preincubated for 2 h with 100 nm concanamycin A (CMA) (Sigma). Cells were analyzed on a FACScalibur flow cytometer, and data were processed using Cell Quest software (BD Biosciences). Cytotoxicity Assay—Cytotoxicity assays were performed using a standard 4-h chromium release assay. Briefly, 2 × 103 51Cr-labeled T1 target cells were incubated for 4 h at 37 °C with effector cells (LT12) at different effector/target ratios in a final volume of 200 μl in 96-well microplates. Experiments were performed in triplicate. At the end of the incubation 40 μl of the supernatant was transferred into 96-well Luminaplate solid scintillation plates (Packard Instrument Co.) and, after overnight drying, counted in a Top Count β counter (Packard). Data were expressed as the percentage of specific lysis at the T1/LT12 cell ratio indicated. The percentage of specific 51Cr release (specific lysis of target cells) was calculated as (experimental release - spontaneous release)/(total release - spontaneous release) × 100. Lytic units present in 107 effector cells were then assessed according to Pross et al. (25Pross H.F. Baines M.G. Rubin P. Shragge P. Patterson M.S. J. Clin. Immunol. 1981; 1: 51-63Crossref PubMed Scopus (681) Google Scholar) using a computer program. One lytic unit was defined as the number of effector cells required for 30% lysis of 3 × 103 target cells. Western Blot Analysis—Total cellular extracts were prepared by lysing cells in ice-cold buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). Equivalent protein extracts (30–50 μg) were denatured by boiling in SDS and β-mercaptoethanol, separated by SDS-PAGE, and transferred onto Hybond™ membranes (Amersham Biosciences). The efficiency of the electrotransfer was assessed by Ponceau Red staining. Blots were blocked overnight with Tris-buffered saline containing 5% nonfat dry milk and probed with appropriated antibody for 1 h (anti-p53 (DO-1), anti-Mdm2 (SMP14), anti-Bid (FL195), and actin (C11)) or overnight (anti-caspase 3 (8G10), anti-phospho-p53, anti-phospho-ATM, and anti-phospho-p38K). After washing, blots were incubated with appropriate horseradish peroxidase-conjugate secondary Ab. The complexes were detected using SuperSignal® West Pico Chemiluminescent Substrate (Pierce). Inhibition of p53 Activation—p53 activation was inhibited by preincubating the T1 tumor cell line with 20 μm pifithrin-α (PFT-α) (BIOMOL Research Laboratories Inc., Plymouth, PA) for 48 h before co-culturing T1 and LT12 CTL clone or before loading with granzyme B. Small Interference RNA (siRNA) Transfection—Two sequences of p53 siRNAs were used. The first siRNA used was designed with the Sigma-Proligo RNA interference designer tool: (siRNA p53_2) 5′-GUG AGC GCU UCG AGA UGU UdTdT, and the second sequence was purchased from Santa Cruz Biotechnology (NM_000546.2) (siRNA p53_JT) CGG CAU GAA CCG GAG GCC CAU dTdT. Subconfluent cells were transfected with siRNA in Opti-MEM I according to the manufacturer's instructions. Confocal Scanning Immunofluorescence Microscopy—T1 cells cultured on coverslips were co-incubated with LT12 CTL clone (effector/target ratio 2/1) for 10, 30, or 60 min. Cells were washed with PBS, fixed with paraformaldehyde (4% w/v in PBS) for 1 h, and then permeabilized with SDS (0.1% w/v in PBS) for 10 min. Nonspecific sites were blocked with fetal bovine serum 10% in PBS for 20 min before staining with anti-p53 (DO-1) monoclonal Ab; p53 expression was detected by Alexa Fluor 546 (red) goat anti-mouse secondary Ab (Molecular Probes). Nuclear staining was performed with TO-PRO®-3 (blue) (Molecular Probes). The coverslips were mounted on glass slides using a drop of Vectashield hard set (Vector Laboratories Inc., Burlingame, CA). The fluorescence was examined under an LSM 510 confocal microscope (Zeiss, Jena, Germany) as previously described (26Abouzahr S. Bismuth G. Gaudin C. Caroll O. Endert VanP. Jalil A. Dausset J. Vergnon I. Richon C. Kauffmann A. Galon J. Raposo G. Mami-Chouaib F. Chouaib S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1428-1433Crossref PubMed Scopus (48) Google Scholar). Loading of Granzyme B—T1 cells were plated on coverslips at a density of 1.5 × 105 cells per well in 6-well plates. Forty-eight hours later cells were loaded with recombinant granzyme B using sublytic doses of the pore-forming protein streptolysin-O (SLO) (Sigma, MDL number MFCD00132389). Briefly, SLO was preactivated by incubating for 30 min at room temperature in PBS containing 1 mm dithiothreitol. Cells were then washed in serum-free medium followed by dropwise addition of 150 μl of RPMI containing 100 nm GrB to cell monolayers. Wells were flooded 15 min later with 1.5 ml of RPMI with 5% fetal bovine serum. Early apoptotic events were evaluated 30 min and 1, 2, or 4 h after loading GrB with Dioc6(3) and propidium iodide labeling. The T1 Target Apoptotic Cell Death Induced by LT12 CTL Clone Is Mediated by the Perforin/Granzymes Pathway—Initially we investigated the susceptibility of T1, the wild type p53 target cells, to the cytotoxicity induced by the autologous CTL clone LT12 using Dioc6(3) and propidium iodide labeling. As shown in Fig. 1, at an effector/target ratio of 2/1, LT12 induced between 30 and 35% apoptotic T1 cells by the times indicated. Incubation of these cells with staurosporin (1 μm for 3 h) used as a positive control resulted in the induction of 50% of apoptotic cells. Moreover, preincubation of T1 target cells with CMA, an inhibitor of cytotoxic granules exocytosis by chelating free calcium, resulted in cell death being dramatically inhibited, indicating that the apoptotic death observed is mediated by the perforin/granzymes pathway. p53 Accumulation in T1 Target Cells after Their Interaction with the LT12 CTL Clone—To gain more insights into p53 implication in the control of CTL-mediated lysis, we asked whether tumor T1 cell interaction with the CTL LT12 clone constitutes cellular stress sufficient to induce p53 activation in tumor cells. To this end we co-incubated T1 tumor target cells with LT12 CTL clone at an effector/target ratio of 2/1 for 10, 30, or 60 min. Western blot analysis consistently revealed that although low level expression of p53 was maintained in non-stressed T1 control target cells, T1/LT12 conjugation resulted in rapid p53 accumulation in T1 tumor target cells (Fig. 2A). Using another melanoma cell line and its specific CTL clone, we obtained data confirming the p53 accumulation in target cells (supplemental Fig. S1). Because p53 activity depends on its expression level and its cell localization, p53 expression in T1 target cells was examined by confocal laser scanning microscopy. Data provided in Fig. 2B show rapid nuclear and cytoplasm p53 accumulation in T1 target cells after CTL-target conjugation. These results underline that CTL hitting of T1 target cells effectively represents significant stress sufficient to induce p53 accumulation. To determine the possible involvement of the cytotoxic granules exocytosis-dependent pathway in cytoplasmic and nuclear p53 accumulation and activity after T1 recognition by LT12, we inhibited the perforin/granzyme-mediated pathway using CMA. Western blot (Fig. 2C) or confocal microscopy (Fig. 2D) analysis showed that preincubation of the LT12 CTL clone with CMA resulted in the inhibition of cytoplasmic and nuclear p53 accumulation and activity at the time indicated. These data show that p53 accumulation and activation observed after T1/LT12 interaction is induced by the perforin/granzymes pathway, further supporting the notion that it constitutes an effective stress in target cells. The Inhibition of p53 Activity or Lowering of Its Expression Induced a Decrease in LT12-mediated T1 Target Lysis—To determine whether the cytoplasmic and nuclear p53 accumulation observed and its activation were involved in LT12-mediated lysis, we performed experiments to inhibit either p53 expression using gene silencing or its activation using PFT-α (27Gorgoulis V.G. Zacharatos P. Kotsinas A. Kletsas D. Mariatos G. Zoumpourlis V. Ryan K.M. Kittas C. Papavassiliou A.G. EMBO J. 2003; 22: 1567-1578Crossref PubMed Scopus (78) Google Scholar). T1 cells were transfected with siRNA targeting the p53 gene. As shown in Fig. 3A, Western blot analyses indicate that both p53 siRNA (siRNA 2 and siRNA JT) were effective in significantly lowering the p53 level (70%), whereas siRNA control (siRNA Sc, a non-targeting siRNA) had only a marginal effect. We then used the p53 inhibitor PFT-α that has been reported to inhibit, in vitro, p53-dependent gene transcription and to protect against a variety of genotoxic agents. As shown in Fig. 3B, when T1 tumor target cell line was preincubated with PFT-α before co-culture with the LT12 CTL clone, inhibition of target lysis (60% inhibition) by LT12 was observed. Furthermore, siRNA targeting p53 was also accompanied by dramatic inhibition of T1 lysis, whereas the siRNA Sc had only a slight effect. Granzyme B-dependent Induction of p53 Phosphorylation and Accumulation in Tumor Target Cells by Granzyme B—Granule-mediated killing by cytotoxic T lymphocytes requires the combined action of the membranolytic protein perforin and granule-associated granzymes. Because the bacterial pore-forming toxin SLO was reported to have the same membranolytic and/or endosome disrupting properties as perforin, it was used in the course of these studies (28Browne K.A. Blink E. Sutton V.R. Froelich C.J. Jans D.A. Trapani J.A. Mol. Cell. Biol. 1999; 19: 8604-8615Crossref PubMed Scopus (178) Google Scholar). To examine the effect of the perforin/granzyme B pathway on the induction of apoptosis in T1 cells, these cells were incubated with sublytic doses of SLO (2 μg/ml) alone or in combination with human recombinant GrB (100 nm). Data shown in Fig. 4A indicate that SLO/GrB combination resulted in strong induction of apoptotic cell death, whereas SLO alone had only a slight effect. The data produced using Western blot analysis and depicted in Fig. 4B indicate that SLO/GrB-induced apoptotic T1 cell death correlates with Bid and caspase 3 cleavage, whereas incubation of T1 cells with SLO alone has no effect on either events (Fig. 4B). To investigate the capability of GrB for inducing p53 accumulation during CTL/target tumor interaction, T1 cells were treated with SLO (2 μg/ml) alone or in combination with recombinant GrB at the times indicated. The Western blot data shown in Fig. 5A indicate that SLO/GrB treatment resulted in the induction of p53 accumulation and transcriptional activity (data not shown). This accumulation was associated with the induction of apoptotic cell death in T1 cells (Fig. 4A). It should be noted, however, that treatment of T1 tumor cells with SLO alone resulted in a less significant increase in p53 that was not accompanied by induction of apoptotic cell death (see Fig. 4A). Given that post-translational modifications such as serine and threonine phosphorylation are fundamental for p53 activation, we asked whether the concomitant SLO/GrB exposure interferes with p53 phosphorylation. Western blot analyses were performed using specific antibodies to evaluate the seven sites of p53 most commonly phosphorylated. Although no phosphorylation was detected in whole cell lysates of control cultures and in cells treated with SLO alone, specific bands corresponding only to Ser-15 and Ser-37 could be observed early on at 30 min and 1 and 2 h after exposure of T1 cells to SLO/GrB (Fig. 5B). These observations suggest that GrB can effectively induce p53 activation at least in part by a mechanism involving Ser-37 and Ser-15 phosphorylation after stress kinase activation. Given that ATM and p38K are involved in the phosphorylation of Ser-15 and Ser-37, respectively, we wondered whether SLO/GrB interfere with the activation of these stress kinases. The results illustrated in Fig. 5C indicate that whereas SLO alone had no effect, the SLO/GrB combination leads to the specific phosphorylation of ATM and p38K in T1 cells at 30 min and 1 and 2 h. Granzyme B-mediated Target Cell Death Involves p53 Phosphorylation—Because p53 phosphorylation is essential in the regulation of its activity and to further investigate how p53 activity impacts on GrB-induced killing of T1 cells, we examined the relationship between p53 phosphorylation and GrB-induced apoptotic cell death. In this aim, to explore this, we preincubated T1 cells with the specific p53 inhibitor PFT-α (20 μm) for 48 h. Data shown in Fig. 6A indicate that such treatment resulted in a significant decrease in GrB-induced apoptosis of T1 cells, which correlated with inhibition of Bid and caspase 3 cleavage (Fig. 6B). These data were confirmed using another melanoma cell line (supplemental Figs. 2 and 3). More interestingly, as depicted in Fig. 6C, the latter event correlated with inhibition of GrB-induced p53 phosphorylation at residues Ser-15 and Ser-37 especially at 1 and 2 h. These observations indicate that GrB-induced phosphorylation of p53 is a key event in coordinating the magnitude of apoptotic target killing. siRNA Targeting p53 Induced Inhibition of SLO/GrB-mediated Apoptotic T1 Cell Death—To investigate the functional consequence of p53 silencing on SLO/GrB-induced apoptotic cell death in T1 cells, p53 was silenced in these cells by RNA interference. As shown in Fig. 7A, small interfering RNA against wild type p53 dramatically depressed p53 expression in T1 cells. We next examined the effect of SLO/GrB on the viability of siRNA-treated cells. Data depicted in Fig. 7B indicate a significant down-regulation in the percentage of apoptotic T1 cells induced by the SLO/GrB treatment. Approaches to treatment of cancer based on the immune system have often focused on specific cytolytic effector cells such as CTLs (29Barry M. Bleackley R.C. Nat. Rev. Immunol. 2002; 2: 401-409Crossref PubMed Google Scholar). The present study was intended to provide insight into the functional relationship between GrB and p53 during tumor-specific lysis mediated by CTLs. Several phenomenological studies on T cell-mediated cytotoxicity in vitro have been extensively reviewed (30Russell J.H. Ley T.J. Annu. Rev. Immunol. 2002; 20: 323-370Crossref PubMed Scopus (824) Google Scholar), and major advances have been made in understanding CTL-mediated apoptosis. However, increasing evidence from studies in patients and on cultured cells has highlighted the possibility that the induction of CTLs may be essential but not sufficient to the control of tumor progression (31Anichini A. Molla A. Mortarini R. Tragni G. Bersani I. Di Nicola M. Gianni A.M. Pilotti S. Dunbar R. Cerundolo V. Parmiani G. J. Exp. Med. 1999; 190: 651-667Crossref PubMed Scopus (165) Google Scholar). It is assumed that tumor cell growth in vivo is influenced not only by the ability of CTLs to recognize and respond to the tumor but also by the susceptibility of tumor cells to host-mediated anti-tumor immune responses (32Medema J.P. de Jong J. van Hall T. Melief C.J. Offringa R. J. Exp. Med. 1999; 190: 1033-1038Crossref PubMed Scopus (306) Google Scholar). Such susceptibility involves not only the effector and target cell features but also their reciprocal interaction, which so far remains not clearly understood. Recently we provided evidence indicating that tumor killing by autologous CTLs can be enhanced by targeting degranulation-independent mechanisms via restoration of wtp53, a key determinant of apoptotic machinery regulation (22Thiery J. Dorothee G. Haddada H. Echchakir H. Richon C. Stancou R. Vergnon I. Benard J. Mami-Chouaib F. Cho" @default.
• W2020838087 created "2016-06-24" @default.
• W2020838087 creator A5020021823 @default.
• W2020838087 creator A5045530749 @default.
• W2020838087 creator A5046508788 @default.
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• W2020838087 creator A5078470028 @default.
• W2020838087 date "2007-11-01" @default.
• W2020838087 modified "2023-10-17" @default.
• W2020838087 title "Granzyme B-induced Cell Death Involves Induction of p53 Tumor Suppressor Gene and Its Activation in Tumor Target Cells" @default.
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