The protective effect of phorbol esters on Fas-mediated ... - Nature

Oncogene (2002) 21, 4957 – 4968 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

The protective effect of phorbol esters on Fas-mediated apoptosis in T cells. Transcriptional and postranscriptional regulation Magali Herrant1, Fre´de´ric Luciano1, Agne`s Loubat2 and Patrick Auberger*,1 1

INSERM U 526, Equipe labellise´e par la Ligue Nationale contre le Cancer, IFR 50, Faculte´ de Me´decine, Avenue de Valombrose, 06107 Nice Cedex 2, France; 2INSERM U364, IFR50, Faculte´ de Me´decine, Avenue de Valombrose, 06107 Nice cedex 2, France

Phorbol esters are tumor promoters that bind and activate both conventional and new Protein kinase C (PKC) isoforms. In various circumstances, PKC-dependent signaling pathways can promote cell survival and protect against cell death. This was first analysed in Jurkat T cells where Phorbol Myristate Acetate (PMA) was found to inhibit Fas-mediated apoptosis as judged by DiOC6(3) staining, caspase activation and DNA fragmentation, indicating that PMA exerts its protective effect upstream or at the mitochondrial level in these cells. PMA activated most of the main kinase pathways in T cells such as PKCs, p42/44MAPK, p38MAPK and p90Rsk but not JNK and Akt. A pharmacological approach allowed us to identify that nPKCs are both necessary and likely sufficient to promote T cell survival. Besides this post-transcriptional regulation, nPKCs may also regulate apoptosis at the transcriptional level. cDNA arrays were used to identify a set of genes whose expression was modulated in death versus survival conditions. Following PMA treatment, expression of Mcl-1 and Bcl-x increased while that of cMyc was significantly reduced. Moreover, survivin expression decreased upon CH11 or PMA treatment. cMyc, survivin and Bcl-x modulation seems to be regulated at the transcriptional level while decrease in Mcl-1 protein in CH11-treated cells resulted especially from a caspasedependent proteolysis. Taken together, our data demonstrate that PMA-mediated inhibition of apoptosis is a complex process that is integrated at both the transcriptional and post-transcriptional level and point out to the potential role of Mcl-1, Bcl-x, c-Myc and survivin in this process. Oncogene (2002) 21, 4957 – 4968. doi:10.1038/sj.onc. 1205689 Keywords: apoptosis; phorbol esters; cDNA arrays; death and survival genes

Introduction Apoptosis is an active form of cell death that plays a fundamental role in normal development, tissue home-

*Correspondence: P Auberger; E-mail: [email protected] Received 21 February 2002; revised 13 May 2002; accepted 20 May 2002

ostasis and pathological situations (Los et al., 1999; Nagata, 1998; Thompson, 1995). Apoptosis induced by ligation of the Fas/CD95 receptor plays a pivotal role in the physiological elimination and turnover of lymphoid cells (Chan et al., 2000; Krammer, 2000; Nagata and Golstein, 1995; Santamaria, 2001). Fas is constitutively expressed in a large majority of T cell lines, in antigen-stimulated T cells from peripheral blood and thymocytes (Alderson et al., 1995; Brunner et al., 1995; Ju et al., 1995). Activation of CD95 by Fas ligand or anti-Fas antibody induces clustering and trimerization of the receptor, followed by its interaction with an adaptator protein called Fas associated protein with death domain (FADD) and subsequent recruitment of caspase-8 a cysteine proteinase to the Fas receptor complex, also known as the deathinducing signaling complex (DISC) (Fesik, 2000; Kischkel et al., 1995). The formation of the DISC initiates the activation of the caspase pathway ultimately leading to cell death. However, in the leukemic T cell line Jurkat, a Fas type II cell, a small amount of caspase 8 is recruited to the DISC upon receptor triggering (Scaffidi et al., 1998). Activated caspase 8, in turn, mediates downstream apoptotic events partly through the cleavage and relocation of the BH3 domain containing proapoptotic Bcl2 family member Bid. While Bid is localized in the cytosol of non-apoptotic cells, truncated Bid (t-Bid) translocates to mitochondria, where it induces release of cytochrome c independently of caspases (Green, 1998, 2000; Li et al., 1998; Zamzami et al., 2000). Phorbol esters are tumor promoters that bind and activate protein kinase C (PKC) isoforms. PKC is one of the critical components of the T cell activation process (Bi et al., 2001; Liu et al., 2000). The PKC family comprised a dozen isoforms that have been classified in three groups according to their structure and cofactor requirements: conventional PKCs (PKCa, PKCb and PKCg) are diacylglycerol- and calciumdependent, novel PKCs (PKC, PKCe, PKCd, PKCy and PKCu) are diacylglycerol dependent but calciumindependent, and finally, atypical PKCs (PKCz, PKCt and PKCl) can bind diacylglycerol but are not activated by phorbol esters (Mellor and Parker, 1998; Mochly-Rosen and Gordon, 1998). In various circumstances, PKC-dependent signaling pathways can promote cell survival and protect against cell death (Gomez-Angelats and Cidlowski, 2001). Overexpres-

Mechanisms of PMA protection against FAS-mediated apoptosis M Herrant et al

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sion of PKCa, e and t increases the resistance of cells to apoptosis and PKC inhibitors are known to sensitize cells to cell death (Bertolotto et al., 2000a; Gray et al., 1997; Whelan and Parker, 1998). Additionally, Fasmediated apoptosis in T cells resulted in a blockade of PKC activity, suggesting a link between these two events (Chen and Faller, 1999). Although involvement of PKCs in the suppression of apoptosis has been reported, only recently have the mechanisms of PMA-mediated protection been analysed. Thus, the MAPK pathway has been implicated in preventing the early and late phase features of CD95-mediated apoptosis in T cells (Holmstrom et al., 1998), but MAPK-independent pathways have also been reported (Tan et al., 1999). For example p90Rsk blocks Bad-mediated cell death via a PKC-dependent pathway that involved new PKC isoforms (Bertolotto et al., 2000a; Tan et al., 1999; Villalba et al., 2001). Alternatively, other mechanisms such as PKC-mediated regulation of CD95 oligomerization and altered formation of the DISC have been evoked (Ruiz-Ruiz et al., 1999). Despite these well documented effects, the mechanisms by which phorbol esters inhibit apoptosis are presently not fully understood. In the present study, we have undertaken experiments to analyse the molecular events underlying PMA-induced inhibition of apoptosis. We show here that PMA activated most of the main survival pathways in T cells, but nPKCs were both necessary and likely sufficient to promote T cell survival. Besides this post-transcriptional regulation, nPKCs also regulate apoptosis at the transcriptional level. In this line, using cDNA arrays we identified of a set of genes including Mcl-1, Bcl-x, survivin and c-Myc, whose expression was modulated in death versus survival conditions. Mcl-1 expression was also found to be regulated at the post-transcriptional level by a caspasedependent cleavage. Taken together, our data indicate that phorbol ester-mediated inhibition of apoptosis is a complex process that involves both transcriptional and post-transcriptional controls and highlight the potential role of Mcl-1, survivin and c-Myc, in the regulation of cell death and survival in T cells. Results Phorbol esters protect T lymphocytes from Fas-mediated apoptosis Exposure of Jurkat T cells to 20 ng/ml PMA induced an inhibition of Fas-mediated apoptosis. While characteristic internucleosomal DNA fragmentation was observed in Jurkat T cells treated with 50 ng/ml CH11, little DNA ladder was detected when cells were cocultured in the presence of CH11 and PMA (Figure 1a). Apoptosis of Jurkat T cells was confirmed by flow cytometric analysis of cells stained with DiOC6(3) and propidium iodide. DiOC6(3) staining was used to monitor the disruption of mitochondrial transmembrane potential which constitutes an early and critical event in many apoptotic processes. CH11 (50 ng/ml) Oncogene

induced a decrease of DiOC6(3) staining in more than 63% of Jurkat cells after a 6 h incubation period, an effect totally reversed by PMA (Figure 1b, left). The effect of CH11 exclusively reflected apoptosis, since very few cells with necrotic features (i.e.: stained with propidium iodide) were detected (Figure 1b, right). CH11 treatment of Jurkat T cells also induced a decrease of procaspases 3 and 8 expression at 6 and 24 h as determined by Western blot experiments (Figure 1c and not shown). The decrease in procaspase expression was correlated with the appearance of the active forms of both enzymes (not shown). Activation of procaspases 3 and 8 was inhibited by PMA treatment (Figure 1c lanes 4 and 8). Taken together, these results confirmed and extended the notion that phorbol esters inhibit early and late events associated with Fas-induced apoptosis in Jurkat T cells (Bertolotto et al., 2000a,b; Datta et al., 2000). CH11 did not alter PMA signaling in Jurkat T cells The mechanisms by which phorbol esters protect cells from apoptosis remain only partially understood. It has been proposed that activation of conventional and/ or new PKC isoforms contributed to the antiapoptotic effect of PMA (Gomez-Angelats and Cidlowski, 2001; Meng et al., 2002). To further investigate the molecular events involved in the protective action of PMA on Fas-mediated apoptosis, we analysed its effect on the main signaling pathways in T cells. PMA induced a rapid and sustained phosphorylation of p42/44, p38 MAPK and p90Rsk (Figure 2a – c) but did not affect the phosphorylation status of JNK and Akt (Figure 2d,e). CH11 failed to induce p42/44 MAPK, JNK, p90Rsk and Akt but triggered a long term activation of p38 MAPK (Figure 2a – e). The robust basal phosphorylation of JNK and Akt remains unchanged whatever the stimuli used (Figure 2d,e). In the presence of CH11 and PMA (survival conditions) p38 MAPK phosphorylation profile was found to be the exact superposition of the profile of each stimuli taken separately (Figure 2c). Globally, treatment of Jurkat T cells with CH11 did not interfere with PMA-mediated signal transduction. It is admitted that the effect of phorbol esters are mediated by conventional and new PKC isoforms (Bertolotto et al., 2000a; Villalba et al., 2001). Activation of PKCs required their relocation from the cytoplasm to the plasma membrane. Expression and relocation of most PKC isoforms was visualized in Jurkat T cells treated for short times with CH11, PMA or the combination of both effectors (Figure 3). Jurkat T cells expressed conventional PKC a, b, new PKC d, e, y and atypical PKC l but not conventional PKC g (Figure 3a) in agreement with our previous observation (Mari et al., 1997). Conversely to cPKCs, nPKCs were already detected in the microsomal fraction of Jurkat T cells (Figure 3a). CH11 did not induce relocation of these different PKC isoforms while PMA triggered redistribution of PKCa, PKCb and increased the relocation of PKCd, y and e. As expected PMA failed to provoke atypical PKCl


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Figure 1 PMA inhibits Fas-mediated apoptosis in Jurkat T cells. (a) Internucleosomal DNA fragmentation was visualized following agarose gel electrophoresis. Jurkat T cells were incubated 6 h with 50 ng/ml anti-Fas mAb (CH11) and/or 20 ng/ml PMA. (b) Mitochondrial membrane depolarization was assessed by the loss of DiOC6(3) staining of the mitochondria. Cells incubated 6 h with 50 ng/ml anti-Fas mAb (CH11) and/or 20 ng/ml PMA were stained with DiOC6(3) (50 nM, 15 min, 378C) and propidium iodide. Percentages indicate apoptotic cells. (c) Cells were lyzed and proteins separated by electrophoresis on 10% polyacrylamide gels. Proteins were then blotted on to PVDF membranes which were incubated with either anti-procaspase 3 or anti-procaspase 8 antibodies. Arrows indicate the position of the zymogens for these two caspases

redistribution (Figure 3a). This short time PMAinduced PKC isoforms redistribution was not affected

by CH11. Finally, activated PKCs were only present in the microsomal fraction of PMA stimulated Jurkat T Oncogene


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Figure 2 PMA activates the main survival pathways in T cells. Jurkat T cells were incubated for different times at 378C with 50 ng/ ml anti-Fas mAb (CH11) and/or 20 ng/ml PMA. Cells were then lyzed and proteins separated by electrophoresis on 10% polyacrylamide gels. Proteins were blotted on to PVDF membranes which were incubated with either anti-p42/44 MAPK, anti-p38 MAPK, anti-JNK, anti-p90Rsk or anti-Akt phospho-specific antibodies

Figure 3 Both c and n-PKCs are activated following PMA-treatment of Jurkat cells. Jurkat T cells were incubated for different times at 378C with 50 ng/ml anti-Fas mAb (CH11) and/or 20 ng/ml PMA. Cells lysates were subfractionated in cytoplasmic and microsomal fractions and proteins contained in each fraction were separated by electrophoresis on 10% polyacrylamide gels. Proteins were then blotted on to PVDF membranes which were incubated with either anti-PKC a, b, g, d, e, y, l or anti-pan phospho PKC antibodies

cells as determined by Western blotting with a pan phospho-specific PKC antibody (Figure 3b). The protective effect of PMA is mediated mainly via novel PKC isoforms and is independent of p42/44 MAPK, p38 MAPK, JNK and Akt In view of the results described above we next used a pharmacological approach to determine which signaling pathways were involved in the protective action of PMA. As caspase activation is required for the induction of apoptosis in our model, we performed caspase assays in the presence or the absence of various drugs and effectors used alone or in combination Oncogene

(Figure 4). CH11 treatment of Jurkat T cells increased caspase activity more than 20-fold as assessed by AcDEVD-pNA hydrolysis, an effect inhibited by PMA. Inhibition of CH11-induced caspase activity by PMA was abolished by GF109203X, and inhibitor of both c and nPKCs but only weakly by the cPKC inhibitor Go¨6976. The low but significant levels of caspase activity achieved by Go¨6976 and Uo126 are likely due to (1) inhibition of basal cPKC or MAPK activities respectively or (2) a toxic effect of the drugs. Finally, neither Uo126 a specific inhibitor of MEK, nor Ly 294002 a potent blocker of PI3K were found to reverse the effect of PMA indicating that the protective action of this phorbol ester on Fas-induced caspase activity


4961 Table 1a and b Classification of regulated genes. The lists contain genes that showed over 1.5-fold induction in at least three different experiments in the presence or the absence of CH11 and/or PMA. The ratios of gene expression at 6 or 24 h under stimulation conditions are shown. Each array was performed three times with reproducible results. A typical experiment is shown Fold repression/cDNA, downregulated upon CH11 treatment 6h 73.5/ 14.3.3 Signal transduction factor

Figure 4 nPKCs promote resistance against Fas-mediated apoptosis. Caspase activity was assessed on cell lysates prepared from cells stimulated for 6 h with 50 ng/ml anti-Fas mAb (CH11), 20 ng/ml PMA in the presence or absence of 1 mM GF109203X, 1 mM Go¨6976, 10 mM Uo126, 10 mM LY294002 using 0.2 mM Ac-DEVD-pNA as substrate. Ac-DEVD-pNA hydrolysis was determined in quadruplicate at different times in the presence or absence of 10 mM Ac-DEVD-CHO in order to measure specific caspase activity. Results are expressed as nanomoles of substrates hydrolysed per min and per milligram of proteins and represent the mean of three different determinations

and apoptosis is mediated essentially by nPKCs and is largely independent of cPKCs, p42/44 MAPK, p38 MAPK, PI3K and Akt. cDNA array analysis of death and survival genes We and others have shown previously that nPKCs promote T cell survival partly by a Rsk-dependent phosphorylation and inactivation of Bad (Bertolotto et al., 2000a; Tan et al., 1999; Villalba et al., 2001). Interestingly, the data presented above suggested that a combined effect of PMA at both the transcriptional and post-transcriptional levels cannot be excluded. We thus used cDNA arrays to characterize genes potentially modulated following CH11, PMA or CH11+PMA treatment. Our experimental approach consisted in the comparison of the expression of mRNAs extracted from control, PMA, CH11 (death condition) or CH11+PMA treated Jurkat T cells (survival condition) at 6 or 24 h. Each DNA array experiment was performed at least three times and gave reproducible results. Globally, cDNA arrays identified a dozen genes that were regulated differently in control versus death or survival conditions (Table 1a,b). Expression of eight relevant genes was evaluated semi-quantitatively by RT – PCR. Among them six, namely Bcl-x, Mcl-1, TNF-a, Trail, LT-a and Fas were up-regulated and two c-Myc and survivin were downregulated by PMA (Figure 5a, f – i). CH11 was found to weakly increase TNF-a and Trail expression, whereas it decreased c-Myc and Fas expression or even suppressed survivin and LT-a expression more particularly at 24 h (Figure 5a, a – c, h). The decreased expression of the expected 190 bp fragment of c-Myc upon PMA treatment was accompanied by an increase of a new 500 bp transcript. The exact nature of this transcript is currently under investigation. Interest-

Fold induction/cDNA, upregulated upon PMA treatment 6h 1.5/ TNF-a TNF superfamily 1.7/ Mcl-1 Bcl-2 related 1.8/ 14.3.3 Signal transduction factor 2.3/ Bcl-x Bcl-2 related 2.3/ LT-a TNF superfamily 2.5/ p105 NF-kB1 Transcription factor 2/ TRAIL TNF superfamily 5.4/ IL-2 Ra Cytokine receptor Fold repression/cDNA,downregulated upon PMA treatment 6h 75.3/ c-Myc Transcription factor Fold induction/cDNA, upregulated upon CH11+PMA treatment 6h 1.5/ IL-2Ra Cytokine receptor 1.7/ TNF-a TNF superfamily 2/ Mcl-1 Bcl-2 related 2.3/ TRAIL TNF superfamily 2.4/ p105 NF-kB1 Transcription factor 2.6/ Bcl-x Bcl-2 related 2.9/ LT-a TNF superfamily Fold repression/cDNA, downregulated upon CH11+PMA treatment 6h 75.2/ c-Myc Transcription factor Table 1b Fold repression/cDNA, downregulated upon CH11 treatment 24 h 71.7/ c-Myc Transcription factor 72.2/ Survivin Inhibitor of apoptosis 73/ 14.3.3 Signal transduction factor Fold induction/cDNA, upregulated upon PMA treatment 24 h 1.2/ 1.4/ 1.5/ 2/ 2.9/ 3.1/ 5.4/ 12.3/

Mcl-1 p105 NF-kB1 Bcl-x TRAIL TNF-a Fas IL-2 Ra LT-a

Bcl-2 related Transcription factor Bcl-2 related TNF superfamily TNF superfamily TNF superfamily Cytokine receptor TNF superfamily

Fold repression/cDNA, downregulated upon PMA treatment 24 h 71.6/ Survivin Inhibitor of apoptosis 75/ c-Myc Transcription factor Continued

ingly, there was no significant differences in gene expression in PMA versus PMA+CH11 treated cells, indicating that PMA efficiently protects cells from CH11 effect (Figure 5). Finally, the effect of PMA on gene expression was reversed by GF109203X as illustrated by the inhibition of PMA-induced LT-a Oncogene


4962 Table 1 (continued) Fold induction/cDNA, upregulated upon CH11+PMA treatment 24 h 1.5/ Mcl-1 Bcl-2 related 1.9/ Bcl-x Bcl-2 related 2.2/ Fas TNF superfamily 2.4/ p105 NF-kB1 Transcription factor 4/ TRAIL TNF superfamily 5.4/ TNF-a TNF superfamily 5.7/ IL-2 Ra Cytokine receptor 14.6/ LT-a TNF superfamily Fold repression/cDNA, downregulated upon CH11+PMA treatment 24 h 71.9/ 14.3.3 Signal transduction factor 72.7/ Survivin Inhibitor of apoptosis 79/ c-Myc Transcription factor

and Mcl-1 mRNA increase and c-Myc and survivin mRNA decrease (Figure 5b). Expression of Mcl-1, survivin and c-myc in death versus survival conditions To confirm at the protein level the data obtained by RT – PCR we next evaluated the effect of CH11, PMA or the combination of both effectors on Mcl-1, survivin, c-Myc, cyclin-A and procaspase 3 expression. Procaspase 3 and cyclin A were chosen as positive and negative controls of cleavage during apoptosis respectively. CH11 and PMA induced a decrease in c-Myc expression that was detected 4 – 6 h following the addition of the effectors and a reduction of survivin expression after 16 h, in agreement with their effect on gene expression (Figure 6c). CH11 induced a rapid cleavage of Mcl-1 that was detectable as soon as 2 h after antibody addition. Cleavage of Mcl-1 generated two fragments of 24 and 13 kDa (Figure 6d and not shown). Cleavage of Mcl-1 was abolished by pretreatment of Jurkat T cells with the large spectrum caspase inhibitor Z-VAD-fmk while c-Myc expression was not significantly reduced in identical conditions (not shown). PMA triggered a significant increase in Mcl-1 protein as soon as 2 h after the beginning of treatment (Figure 6d). Mcl-1 expression returned to basal level after 8 – 12 h (not shown), in agreement with the cDNA array results (Table 1a,b). PMA was found to inhibit Fas-mediated Mcl-1 cleavage, an effect which contributes to maintain high levels of the mature protein in survival condition (Figure 6d). Expression of cyclin A, used as a control, remained stable whatever the stimuli (Figure 6b). Finally, CH11 apoptotic effect was monitored by the rapid disappearance of procaspase 3 reflecting its activation (Figure 6a). As expected, cleavage and activation of procaspase 3 was also inhibited in the presence of PMA (Figure 6a). In conclusion, the Western blot experiment depicted in Figure 6 confirmed the results obtained by arrays and RT – PCR and also showed that besides a transcriptional control, Mcl-1 was also regulated by a caspasedependent proteolysis following Fas-triggering. Oncogene

Mcl-1 is cleaved by caspases 3, 6 and 7 in vitro In view of the results previously described we next examined whether Mcl-1 may serve as a substrate for recombinant caspases. In vitro transcribed and translated Mcl-1 and the Src family tyrosine kinase Fyn used as a positive control (Luciano et al., 2001; Ricci et al., 1999) were incubated at 378C with either purified recombinant caspases or cell extracts prepared from control (Ext7) or Fas-stimulated Jurkat T cells (Ext+). As shown on Figure 7, two Mcl-1 fragments were detected in control conditions. After 15 h at 378C, Mcl-1 was proteolytically processed by recombinant caspases 3, 6 and 7 with however a better efficiency for caspases 3 and 6 and by cellular extracts prepared from apoptotic cells, generating the characteristic 24 kDa fragment previously detected in intact cells (Figure 7). Cleavage of Mcl-1 was inhibited by preincubation with the caspase inhibitor Ac-DEVD-CHO. Recombinant caspases 6 and 7 were also shown to induce the characteristic cleavage of p59Fyn (Ricci et al., 1999). When a more reticulated polyacrylamide gel was used (12.5%), two cleavage products were detected for Mcl-1 at 24 and 13 kDa both in vitro and in intact cells (Figure 6, 7 and not shown). Cleavage of Mcl-1 was also detected in thymocytes undergoing apoptosis (not shown). Finally, two cleavage sites were characterized in Mcl-1 after Asp127 and 157. The exact role of Mcl-1 cleavage is currently under investigation (Herrant et al, in preparation). Discussion Molecular mechanisms underlying PMA protection on Fas-mediated apoptosis in Jurkat T cells Although involvement of PKCs in the activation or suppression of apoptosis has been previously reported, (Whelan and Parker, 1998) only recently have the mechanisms of PMA-mediated protection been analysed. They included activation of survival pathways such as p42/44 MAPK, PI3K/Akt, p90Rsk, NFkB or inhibition of the DISC assembly (Bertolotto et al., 2000a; Bonni et al., 1999; Holmstrom et al., 2000; Meng et al., 2002; Ruiz-Ruiz et al., 1999; Tan et al., 1999; Villalba et al., 2001). We and others have shown previously that a p90Rsk-dependent phosphorylation of the proapoptotic protein Bad might contribute at least in part to the protective effect of PMA on Fas or Trail-induced apoptosis in T leukemic cell lines (Bertolotto et al., 2000a; Tan et al., 1999). Indeed, phosphorylated Bad does not bind mitochondrial antiapoptotic members of the Bcl-2 family due to its sequestration in the cytosol by 14.3.3 (Zha et al., 1996). However, despite these well documented effects, the way by which PKCs inhibit apoptosis is not fully understood. To gain further insights into these mechanisms of protection, we analysed the effect of PMA on Fas-mediated apoptosis in the T leukemic cell line Jurkat. In these cells, Fas-induced mitochondrial depolarization, caspase activation and internucleosomal DNA fragmentation were significantly inhibited


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Figure 5 (a) RT – PCR analysis of genes modulated in death and survival conditions. Amplification method was described in Materials and methods. Actin was used as an invariant control in the experiment. Note the presence of two amplified fragments for cMyc: (a) 25 cycles. (b) 30 cycles. (b) The PMA effect on gene modulation is PKC-dependent. Amplification was performed as described in (a)

following PMA-treatment, suggesting that the protective effect of PMA is integrated at least partly upstream or at the mitochondrial level since this phorbol ester can inhibit the Fas-induced decrease in mitochondrial Dcm. This short term inhibition of apoptosis by PMA is in line with recent studies showing that phorbol esters may interfere with DISC formation (Meng et al., 2002; Ruiz-Ruiz et al., 1999) and with our previous results indicating that PMA inhibited death receptor-induced apoptosis in T cell by a Rsk-dependent phosphorylation and sequestration of Bad (Bertolotto et al., 2000a).

cDNA gene profiling of PMA protective effect on apoptosis If post-transcriptional nPKC-dependent mechanisms could explain in part the protective effect of PMA on death receptor-mediated apoptosis in T cells, the possibility of a transcriptional control cannot be excluded. To investigate gene expression events in death versus survival conditions we used a cDNA array approach. We decided to focalize our attention on a restricted panel of 200 highly relevant genes (apoptosis, cytokines and their receptors and cell cycle regulation) Oncogene


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Figure 6 Time-course of Mcl-1, survivin and c-Myc expression in Jurkat T cells. Jurkat T cells were incubated for different times at 378C with 50 ng/ml anti-Fas mAb (CH11) and/or 20 ng/ml PMA. Cells were then lyzed and proteins separated by electrophoresis on 12% polyacrylamide gels. Proteins were then blotted onto PVDF membranes which were incubated with either anti-Procaspase 3, anti-Cyclin A, anti-c-Myc, anti-Mcl-1 or anti-Survivin antibodies. *Indicates the position of the 24 kDa cleaved fragment

Figure 7 Mcl-1 is a new substrate for caspases during Fasmediated apoptosis. Mcl-1 and p59Fyn (as a control) were transcribed and translated in vitro with 35S-methionine and incubated with purified recombinant caspases 3, 6, 7 (25 ng), Fas-activated (Ext+) or non activated (Ext-) Jurkat T-cells extracts (100 mg) for 15 h at 378C in the presence or absence of Ac-DEVD-CHO (10 mM). The reaction products were then analysed by SDS – PAGE and autoradiography. *Indicates the cleaved form of Mcl-1 and Fyn (p57Fyn)

rather than on a larger collection. We analysed expression of genes potentially involved in death (CH11 treatment) versus survival (CH11+PMA treatment) following a short term (6 h) or a long term (24 h) stimulation period. Only minor qualitative and quantitative differences were observed between the two incubation times, the overall panel of genes being very similar in each case (Table 1a,b). Our data indicated the regulation of approximately 10% of the represented genes by a factor 1.5 or higher upon PMA treatment (range 1.5 to 12.5). In cells induced to undergo apoptosis with CH11 only 1 – 2% of the represented genes were found to be modulated (range 71.7 to Oncogene

73.5). The number of PMA regulated genes could seem elevated a priori but is expected since most of the genes present on the array are involved in apoptosis, cell division or encoded cytokines or their receptors. It should be pointed out that several other genes were found to be modulated in death versus survival conditions but were not mentioned in Table 1a,b since their threshold levels were low rendering their modulation difficult to ascertain with precision. Interestingly, modulation of gene expression under the different experimental conditions used for the cDNA arrays was largely confirmed by RT – PCR experiments (Figure 5a). Notably, following PMA treatment, expression of the Bcl2 family members Mcl-1 and Bcl-x increased while that of c-Myc and the IAP, survivin decreased. It is worth mentioning that in the same conditions we also observed increase in TNFa, Trail, LTa and Fas expression. The transcriptional effect of the phorbol ester was PKC-dependent since both PMA-induced increase and decrease in gene expression were abolished in the presence of the PKC inhibitor GF109203X (Figure 5b). Upon Fas triggering, expression of survivin, Myc and Bcl-x were significantly reduced while that of Mcl-1 slightly diminished. Conversely, we failed to detect a decrease in Bcl-x and Mcl-1 expression in our cDNA array analysis. This is not surprising since (1) expression of these particular genes in the cDNA array experiments was low and (2) RT – PCR represents a much more sensitive method than cDNA arrays. Expression and potential role of the characterized proteins in T cell death and survival As shown by RT – PCR c-Myc down-regulation seems to be mainly controlled at the transcriptional level in


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PMA treated cells. The c-Myc proteins are members of a basic region/helix-loop-helix/leucine zipper transcription factor family that binds to and transactivates through consensus CACGTG sequences a large number of cellular targets involved in growth control, differentiation, apoptosis and metabolism (de Alboran et al., 2001; Genestier et al., 1999; Hueber et al., 1997; Kasibhatla et al., 2000; Oberg et al., 2001; Tiberio et al., 2001). Up-regulated c-Myc expression is generally associated to proliferation but numerous studies have linked either up-regulated or down-regulated c-Myc expression to apoptosis. In addition to its ability to stimulate transcription, c-Myc is also able to repress gene expression (Dang, 1999; Nikiforov et al., 2000; Sonenshein, 1997; Wu et al., 1996). In murine and human B cell lymphoma lines for example, apoptosisinduced by surface IgM triggering correlates with down-regulation of c-Myc expression (Fischer et al., 1994). We show here that phorbol esters which rescue cells from Fas-mediated apoptosis induce a nearly complete down-regulation of c-Myc, suggesting that down-regulation of c-Myc may play an important role in PMA-mediated T cell survival. Mcl-1 is a member of the Bcl2 family that is rapidly up-regulated upon exposure of human myeloblastic leukemia to the differentiation-inducing agent PMA (Kozopas et al., 1993; Townsend et al., 1998, 1999). Mcl-1 is known to function as an anti-apoptotic protein and increase in Mcl-1 expression is correlated with cell survival in myeloblastic leukemia cell lines (Liu et al., 2001; Sordet et al., 2001; Zhou et al., 1997). The data presented here show that Mcl-1 expression is also rapidly increased in the T lymphoblastic leukemia Jurkat upon PMA treatment, an effect that also correlates with cell survival. Conversely to the PMAinduced increase in Mcl-1 expression, engagement of the Fas receptor leads to a decrease in Mcl-1 protein. This decrease in Mcl-1 expression was not observed at the mRNA level (at least at 6 h) and is due to a caspase-dependent proteolysis as judged by both in vivo and in vitro experiments. Interestingly, in survival conditions (CH11+PMA) the Fas-dependent cleavage of Mcl-1 was significantly inhibited, contributing to maintain a high level of the mature protein. Finally, cleavage of Mcl-1 was also detected in primary cells i.e. in mouse thymocytes undergoing spontaneous apoptosis at 378C (not shown). This is the first description of a cleavage of Mcl-1 during the onset of apoptosis. The consequence of Mcl-1 cleavage on its anti-apoptotic function is currently under investigation. Nevertheless, it is not surprising that Mcl-1 and Bcl-x, two important anti-apoptotic members of the Bcl2 family, were induced under survival conditions since increased expression of these two molecules are often associated with protection against apoptosis (Bannerman et al., 2001). In death conditions we also found a strong inhibition of survivin expression both at the mRNA and protein levels. Survivin is one of the inhibitor of apoptosis protein family that can block apoptosis by inhibiting caspase activation (Muchmore et al., 2000; O’Connor et al., 2000). To our knowledge, inhibition

of survivin expression upon Fas treatment has not been previously reported but may contribute further to enhance Fas-mediated apoptosis. The mechanism by which Fas-triggering leads to down-regulation of survivin mRNA expression is presently not known but will merit further studies. Taken together our data demonstrate that the protective effect of phorbol esters on death receptormediated apoptosis is a highly regulated process that might be integrated at both the transcriptional and post-transcriptional levels. For example, the expression of Mcl-1 is rapidly and transiently increased by PMA in Jurkat T cells. Conversely, Fas engagement resulted in a rapid decrease in Mcl-1 expression by a caspase dependent-mechanism. In survival conditions (CH11+PMA) the increase in Mcl-1 expression is also accompanied by a PMA-dependent inhibition of Mcl-1 proteolysis. This combined regulation at the transcriptional and post-transcriptional level contributes to maintain the high level of the Mcl-1 protein and to protect cells from apoptosis. Although increased expression of anti-apoptotic genes in survival conditions was speculated, the data presented here point out to a potential role for Mcl-1, survivin, Bcl-x and c-Myc in the protective effect of PMA. Further studies should aim at analysing the precise role of each of these proteins taken individually in the regulation of cell death and survival in hematopoietic cells. Materials and methods Reagents and antibodies The fluorescent dye 3,3’-dihexyloxacarbocyanine iodide (DiOC6(3)) was obtained from Calbiochem. Stock solution (50 mM) in DMSO was stored at 7208C. Working solution (5 mM) in phosphate-buffered saline (PBS) was used at a final concentration of 50 nM. Bisindolylmaleimide-l (GF109203X), Go¨6976, Uo126, LY294002 were from Calbiochem. Phorbol 12-myristate 13-acetate (PMA), sodium fluoride, sodium orthovanadate, phenylmethylsulfonyl fluoride, aprotinin, and leupeptin were purchased from Sigma. RNase and proteinase K were from Roche Molecular Biochemicals. RPMI and fetal calf serum (FCS) were from Life Technologies, Inc. c-Myc, Mcl-1 and Survivin antibodies were purchased from Santa Cruz Biotechnology; anti-Fas monoclonal antibody (CH11) was from Euromedex, anticaspase 8 antibody was from Oncogene Research Products and anti-caspase 3 antibody was from Transduction Laboratories. Phospho-p42/44, phospho-p38, phospho-Akt antibodies were purchased from Cell Signaling Technology and phospho-JNK from BioLabs. Peroxidase-conjugated anti-rabbit, anti-mouse and anti-goat antibodies were from Dakopatts. Cells Jurkat T cells (clone Jd) were grown at 378C under 5% CO2 in RPMI 1640 medium supplemented with 5% FCS and 100 mg/ml penicillin/streptomycin (Mari et al., 1997; Ricci et al., 2001b). Freshly prepared thymocytes from 12-week-old mice were plated in RPMI 1640 medium supplemented with 10% FCS at 4 or 378C under 5% CO2 as previously described (Pages et al., 1999). Oncogene


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Mitochondrial membrane depolarization Jurkat T cells were suspended at a concentration of 106/ml in fresh medium. The cells were exposed to the different effectors and then incubated with 50 nM DiOC6(3) and propidium iodide at 378C for 15 min (Zamzami et al., 1995). The cells were resuspended in PBS containing 2.5 mM MgCl2 and DiOC6(3) and IP fluorescence were measured by using the FL1 and FL2 channels of a FACScan (Becton Dickinson, Cowley, UK). DNA fragmentation Jurkat T cells exposed to the different effectors were collected and lysed in 200 ml of lysis buffer containing 10 mM Tris (pH 7.5), 1 mM EDTA, and 0.2% Triton X-100. Samples were treated with 100 mg/ml RNase for 30 min and then treated with 100 mg/ml proteinase K for 30 min at 378C as described previously (Luciano et al., 2001; Ricci et al., 1999). Cellular DNA was isopropanol-precipitated, dried, resuspended in Tris-EDTA buffer and incubated for 30 min at 558C. DNA was analysed by electrophoresis on 1.4% agarose gels containing ethidium bromide. Western blot assays Jurkat T cells were incubated with different effectors for the times indicated in the figure legends, and then lysed in buffer B containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 20 mM EDTA, 100 mM NaF, 10 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 20 mg/ml aprotinin, and 1% Triton X-100. 100 mg of protein were separated on 8 to 12% polyacrylamide gel, transferred to PVDF membrane (Immobilon, Millipore) (Ricci et al., 2001a). After blocking non-specific binding sites, the membranes were incubated with specific antibodies. The membranes were washed three times with TNA-1% NP40 (Tris 50 mM, NaCl 150 mM, pH 7.5) incubated further with horseradish peroxidase conjugated antibody for 60 min at room temperature. Immunoblots were revealed by autoradiography using the enhanced chemiluminescence detection kit (Amersham). In vitro transcription and translation of Mcl-1 and p59Fyn and cleavage in a cell-free system Mcl-1 and p59Fyn were transcribed and translated using the Promega TNT coupled reticulocyte lysate system in the presence of 35S-methionine as previously described (Bertolotto et al., 2000b). Briefly, 2.5 ml of reticulocyte lysates were incubated in 50 ml of 25 mM HEPES pH 7.5, 0.1% CHAPS, 5 mM DTT with 25 ng recombinant caspases 3, 6, 7 or 100 mg of Fas-stimulated Jurkat T cell extracts for 8 h at 378C. In some experiments, the effect of caspase inhibitor (Ac-DEVD-CHO, 10 mM) were also monitored. Proteins contained in cell lysates were electrophoresed on 11% polyacrylamide gels. Gels were autoradiographed using Amersham hyperfilm. Subcellular fractions After stimulation, Jurkat T cells (4.106) were resuspended in hypotonic buffer containing 100 mM Tris-HCl pH 7.4, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 20 mg/ml aprotinin and rapidly sonicated at 48C. Cell extracts were first centrifuged at 2000 g for 10 min. Supernatants were then centrifuged at 100 000 g for 45 min. Soluble proteins Oncogene

(cytosol) were collected. Pellets (microsomal fraction) were lysed in buffer B. In each lane the amount of total, cytosolic and microsomal proteins loaded on the gel corresponds to 4.106 cells (Ricci et al., 1999). Expression array R&D Systems’ human Apoptosis Expression Array represents a comprehensive collection of genes implicated in apoptosis, including pro- and anti-apoptotic factors, cell cycle regulators, caspases, signal transduction factors, cytokines and their receptors, and other factors involved in apoptosis. The array consists of 198 different cloned cDNAs, printed as PCR products, on a positively charged nylon membrane. Each cDNA is printed in duplicate with 10 ng of DNA per spot. In addition, eight positive control ‘housekeeping’ genes, six negative controls and human genomic DNA are included on the array. Total RNA was isolated using TriPure reagent (Boehringer Mannheim) from cells incubated for 6 or 24 h with the different effectors. CH11 was used at 50 or 10 ng/ml respectively when a 6 or 24 h incubation period was used, while PMA was added at a concentration of 20 ng/ml whatever the time of incubation. These concentrations of CH11 were shown to induce death of approximately 60% of the cells at both times and allowed to isolate comparable amount of RNA at 6 or 24 h (not shown). After purification, the total RNA pellets were resuspended in water and quantified using OD260 spectrophotometry (Perkin Elmer MBA2000). Ribosomal RNA integrity was verified on a 1% agarose gel electrophoresis. Probe synthesis was performed as recommended by the manufacturer with some modifications. The cDNA labeling reaction involves two steps. First, 10 mg of total RNA in a 15 ml reaction with 4 ml Human Apoptosis-specific Primers were heated to 908C for 2 min and then ramped to 428C in 10 min. Second, in a 30 ml reaction, the RNA were incubated at 428C for 3 h with 1 mCi [a-33P]dATP (3000 Ci/mmole), 333 mM dCTP, 333 mM dGTP, 333 mM dTTP, 20 U Rnasin, Reverse Transcriptase Buffer, 50 U Reverse Transcriptase Superscript II (Gibco – BRL). Unincorporated radiolabelled nucleotides were removed with Micro Bio-Spin Chromatography Columns (Biorad) and probe yields were quantified. Probes were hybridized to each array membrane for approximately 15 h in the Hybridization Solution at 658C as recommended by the manufacturer. The array membranes were washed in decreasing concentrations of SSC. The damp arrays were sealed in Saran wrap and exposed to a phosphor-imaging screen for 3 – 5 days. Exposed screens were scanned using a phosphor-imager STORM 840 (Molecular Dynamics). The images were analysed by the Image Quant 5.0 software at the highest available resolution and a database software (Excel, Microsoft) was required for further investigation. Comparison of the signals from two different samples allows identification of differentially expressed mRNAs. The signal from one or more of the housekeeper genes can be used to normalize signals of all genes on one array and the signal from a blank portion of the cDNA was taken as background. Data are presented as a ratio generated by converting signal intensities to a ratio adjusted for background and one or more housekeeper genes expression: ((gene A probe A – background from probe A)/(housekeeper gene probe A – background from probe A))/((gene B probe B – background from probe B)/(housekeeper gene probe B – background from probe B)). Both membranes were hybridized three to four times, stripping and switching membranes between each hybridization.


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RT – PCR analysis To confirm differential mRNA expression, a panel of genes was quantified by RT – PCR. The genes assayed were: TNFa, Trail, Fas, Lymphotoxin-a (LT-a), Mcl-1, c-Myc, Bcl-x and Survivin. Actin was quantified as an internal control. Total RNA were isolated from cells using TriPure reagent (Boehringer Mannheim). Briefly, 5 mg of total RNA was primed with 1 mg of oligo(dT) primer (Gibco – BRL) and then reverse transcribed at 428C for 1 h. The reaction mix (30 ml) also contained 6 ml of 56 reaction buffer, 1 mM dithiothreitol, 1 mM dNTPs (Amersham Pharmacia Biotech Inc), 50 U Reverse Transcriptase Superscript II (Gibco – BRL). After cDNA synthesis the total volume was made up to 200 ml with H2O. Each PCR reaction contained 5 ml of cDNA, 2.5 ml of 106 reaction buffer (Gibco – BRL), 1 ml of 50 mM MgCl2, 0.5 ml of 100 mM dNTPs, 1 ml of 10 mM forward and reverse primer, 0.12 ml (25 U/l) of Taq polymerase (Gibco – BRL) and H2O for a 25 ml total volume. Cycling parameters were 958C for 3 min followed by 20 to 30 cycles of 958C for 1 min, 608C for 1 min, 728C for 1 min, and 728C for 6 min. The primer sequences were: Survivin forward 5’-GCATGGGTGCCCCGACGTTG-3’, reverse 5’-GCTCCGGCCAGAGGCCTCAA-3’ (451 bp); Bcl-x forward 5’-ATGTCTCAGAGCAACCGGGAG-3’, reverse 5’-TCATTTCCGACTGAAGAGTGA-3’ (406. bp); Mcl-1 forward 5’-CGGCGATCGCTGGAGATTAT-3’, reverse 5’-GTGGTGGTGGTTGGTTA-3’ (497 bp); c-Myc forward 5’-ACCACCAGCAGCGACTCTGA-3’, reverse 5’-TCCAGCAGAAGGTGATCCAGACT-3’ (117 bp); TNF-a forward 5’-GAGTGACAAGCC-

TGTAGCCC-3’, reverse 5’-GCAATGATCCCAAAGTAGAC-3’ (444 bp); Trail forward 5’-AGACCTGCGTGCTGATCGTG-3’, reverse 5’-TTATTTTGCGGCCCAGAGCC-3’ (413 bp); Lymphotoxin-a forward 5’-GCTCTCTTCCTCCCAAGGGTG-3’, reverse 5’-GTGGTACATCGAGTGCAGCCAG-3’ (492 bp); Fas forward 5’-AAGAGAAAGGAAGTACAGAAA-3’, reverse 5’-GACCAAGCTTTGGATTTCATT3’ (430 bp). Caspase activity measurement Each assay (in quadruplicate) was performed with 50 mg of protein prepared from control cells or cells stimulated for 4 h with 50 ng/ml CH11, 20 ng/ml PMA, 1 mM GF109203X, 1 mM Go¨6976, 10 mM Uo126, 10 mM LY294002 as described previously (Ricci et al., 2001b). Briefly, cellular extracts were then incubated in a 96-well plate, with 0.2 mM of Ac-DEVDpNA as substrate for various times at 378C. Caspase activity was measured at 410 nm in the presence or absence of 1 mM of Ac-DEVD-CHO. The specific caspase activity was expressed in nmoles of paranitroaniline released per min and per mg of protein.

Acknowledgments This work was supported by INSERM and the Ligue Nationale Contre le Cancer. We thank Dr R Craig and Pr E Solary for the kind gift of the Mcl-1 constructs and Dr G Ponzio for the anti-cyclin A antibody.

References Alderson MR, Tough TW, Davis-Smith T, Braddy S, Falk B, Schooley KA, Goodwin RG, Smith CA, Ramsdell F and Lynch DH. (1995). J. Exp. Med., 181, 71 – 77. Bannerman DD, Tupper JC, Ricketts WA, Bennett CF, Winn RK and Harlan JM. (2001). J. Biol. Chem., 276, 14924 – 14932. Bertolotto C, Maulon L, Filippa N, Baier G and Auberger P. (2000a). J. Biol. Chem., 275, 37246 – 37250. Bertolotto C, Ricci JE, Luciano F, Mari B, Chambard JC and Auberger P. (2000b). J. Biol. Chem., 275, 37246 – 37250. Bi K, Tanaka Y, Coudronniere N, Sugie K, Hong S, van Stipdonk MJ and Altman A. (2001). Nat. Immunol., 2, 556 – 563. Bonni A, Brunet A, West AE, Datta SR, Takasu MA and Greenberg ME. (1999). Science, 286, 1358 – 1362. Brunner T, Mogil RJ, LaFace D, Yoo NJ, Mahboubi A, Echeverri F, Martin SJ, Force WR, Lynch DH, Ware CF and Green DR. (1995). Nature, 373, 441 – 444. Chan KF, Siegel MR and Lenardo JM. (2000). Immunity, 13, 419 – 422. Chen CY and Faller DV. (1999). J. Biol. Chem., 274, 15320 – 15328. Dang CV. (1999). Mol. Cell. Biol., 19, 1 – 11. Datta R, Yoshinaga K, Kaneki M, Pandey P and Kufe D. (2000). J. Biol. Chem., 275, 41000 – 41003. de Alboran IM, O’Hagan RC, Gartner F, Malynn B, Davidson L, Rickert R, Rajewsky K, DePinho RA and Alt FW. (2001). Immunity, 14, 45 – 55. Fesik SW. (2000). Cell, 103, 273 – 282. Fischer G, Kent SC, Joseph L, Green DR and Scott DW. (1994). J. Exp. Med., 179, 221 – 228.

Genestier L, Kasibhatla S, Brunner T and Green DR. (1999). J. Exp. Med., 189, 231 – 239. Gomez-Angelats M and Cidlowski JA. (2001). J. Biol. Chem., 276, 44944 – 44952. Gray MO, Karliner JS and Mochly-Rosen D. (1997). J. Biol. Chem., 272, 30945 – 30951. Green DR. (1998). Cell, 94, 695 – 698. Green DR. (2000). Cell, 102, 1 – 4. Holmstrom TH, Chow SC, Elo I, Coffey ET, Orrenius S, Sistonen L and Eriksson JE. (1998). J. Immunol., 160, 2626 – 2636. Holmstrom TH, Schmitz I, Soderstrom TS, Poukkula M, Johnson VL, Chow SC, Krammer PH and Eriksson JE. (2000). EMBO J., 19, 5418 – 5428. Hueber AO, Zornig M, Lyon D, Suda T, Nagata S and Evan GI. (1997). Science, 278, 1305 – 1309. Ju ST, Panka DJ, Cui H, Ettinger R, el-Khatib M, Sherr DH, Stanger BZ and Marshak-Rothstein A. (1995). Nature, 373, 444 – 448. Kasibhatla S, Beere HM, Brunner T, Echeverri F and Green DR. (2000). Curr. Biol., 10, 1205 – 1208. Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M, Krammer PH and Peter ME. (1995). EMBO J., 14, 5579 – 5588. Kozopas KM, Yang T, Buchan HL, Zhou P and Craig RW. (1993). Proc. Natl. Acad. Sci. USA, 90, 3516 – 3520. Krammer PH. (2000). Nature, 407, 789 – 795. Li H, Zhu H, Xu CJ and Yuan J. (1998). Cell, 94, 491 – 501. Liu H, Perlman H, Pagliari LJ and Pope RM. (2001). J. Exp. Med., 194, 113 – 126. Liu Y, Witte S, Liu YC, Doyle M, Elly C and Altman A. (2000). J. Biol. Chem., 275, 3603 – 3609.

Oncogene


4968

Los M, Wesselborg S and Schulze-Osthoff K. (1999). Immunity, 10, 629 – 639. Luciano F, Ricci JE and Auberger P. (2001). Oncogene, 20, 4935 – 4941. Mari B, Guerin S, Maulon L, Belhacene N, Farahi Far D, Imbert V, Rossi B, Peyron JF and Auberger P. (1997). FASEB J., 11, 869 – 879. Mellor H and Parker PJ. (1998). Biochem. J., 332, 281 – 292. Meng XW, Heldebrant MP and Kaufmann SH. (2002). J. Biol. Chem., 277, 3776 – 3783. Mochly-Rosen D and Gordon AS. (1998). FASEB J., 12, 35 – 42. Muchmore SW, Chen J, Jakob C, Zakula D, Matayoshi ED, Wu W, Zhang H, Li F, Ng SC and Altieri DC. (2000). Mol. Cell, 6, 173 – 182. Nagata S. (1998). J. Hum. Genet., 43, 2 – 8. Nagata S and Golstein P. (1995). Science, 267, 1449 – 1456. Nikiforov MA, Kotenko I, Petrenko O, Beavis A, Valenick L, Lemischka I and Cole MD. (2000). Oncogene, 19, 4828 – 4831. O’Connor DS, Schechner JS, Adida C, Mesri M, Rothermel AL, Li F, Nath AK, Pober JS and Altieri DC. (2000). Am. J. Pathol., 156, 393 – 398. Oberg F, Wu S, Bahram F, Nilsson K and Larsson LG. (2001). Leukemia, 15, 217 – 227. Pages G, Guerin S, Grall D, Bonino F, Smith A, Anjuere F, Auberger P and Pouyssegur J. (1999). Science, 286, 1374 – 1377. Ricci JE, Lang V, Luciano F, Belhacene N, Giordanengo V, Michel F, Bismuth G and Auberger P. (2001a). FASEB J., 15, 1777 – 1779. Ricci JE, Maulon L, Battaglione-Hofman V, Bertolotto C, Luciano F, Mari B, Hofman P and Auberger P. (2001b). Eur. Cytokine Netw., 12, 126 – 134. Ricci JE, Maulon L, Luciano F, Guerin S, Livolsi A, Mari B, Breittmayer JP, Peyron JF and Auberger P. (1999). Oncogene, 18, 3963 – 3969. Ruiz-Ruiz C, Robledo G, Font J, Izquierdo M and LopezRivas A. (1999). J. Immunol., 163, 4737 – 4746.

Oncogene

Santamaria P. (2001). Curr. Opin. Immunol., 13, 663 – 669. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH and Peter ME. (1998). EMBO J., 17, 1675 – 1687. Sonenshein GE. (1997). J. Immunol., 158, 1994 – 1997. Sordet O, Rebe C, Leroy I, Bruey JM, Garrido C, Miguet C, Lizard G, Plenchette S, Corcos L and Solary E. (2001). Blood, 97, 3931 – 3940. Tan Y, Ruan H, Demeter MR and Comb MJ. (1999). J. Biol. Chem., 274, 34859 – 34867. Thompson CB. (1995). Science, 267, 1456 – 1462. Tiberio L, Maier JA and Schiaffonati L. (2001). Cell Death Differ., 8, 967 – 976. Townsend KJ, Trusty JL, Traupman MA, Eastman A and Craig RW. (1998). Oncogene, 17, 1223 – 1234. Townsend KJ, Zhou P, Qian L, Bieszczad CK, Lowrey CH, Yen A and Craig RW. (1999). J. Biol. Chem., 274, 1801 – 1813. Villalba M, Bushway P and Altman A. (2001). J. Immunol., 166, 5955 – 5963. Whelan RD and Parker PJ. (1998). Oncogene, 16, 1939 – 1944. Wu M, Arsura M, Bellas RE, FitzGerald MJ, Lee H, Schauer SL, Sherr DH and Sonenshein GE. (1996). Mol. Cell. Biol., 16, 5015 – 5025. Zamzami N, El Hamel C, Maisse C, Brenner C, MunozPinedo C, Belzacq AS, Costantini P, Vieira H, Loeffler M, Molle G and Kroemer G. (2000). Oncogene, 19, 6342 – 6350. Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssiere JL, Petit PX and Kroemer G. (1995). J. Exp. Med., 181, 1661 – 1672. Zha J, Harada H, Yang E, Jockel J and Korsmeyer SJ. (1996). Cell, 87, 619 – 628. Zhou P, Qian L, Kozopas KM and Craig RW. (1997). Blood, 89, 630 – 643.