Caroline Salon

Béatrice Eymin

Olivier Micheau

Laurence Chaperot

Joël Plumas

Christian Brambilla

Elisabeth Brambilla

Sylvie Gazzeri

Béatrice Eymin

Dr Beatrice Eymin
email: beatrice.eymin@ujf-grenoble.fr

E2F1 induces apoptosis and sensitizes human lung adenocarcinoma cells to death-receptor-mediated apoptosis through specific downregulation of c-FLIP(short)

Keywords: Apoptosis, Death Receptor, E2F1, c-FLIP, Lung tumors APAF-1, Apoptosis Protease-Activating Factor 1, FADD, Fas-Associated Death Domain-containing protein, FasL, Fas Ligand, FLICE, Fas-associated death domain-Like Interleukin 1β-Converting Enzyme, DISC, Death-Inducing Signaling Complex, c-FLIP, FLICE Inhibitory Protein, RB, Retinoblastoma, TRAIL, Tumor necrosis Factor-Related Apoptosis-Inducing Ligand

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Introduction

The E2F transcription factors family plays a major role in regulating a diverse array of cellular functions including proliferation, differentiation and apoptosis [START_REF] Degregori | The genetics of the E2F family of transcription factors: shared functions and unique roles[END_REF]. So far, seven distinct E2F proteins (E2F1 to 7) with different affinities for the pRB pocket family have been identified [START_REF] Dyson | The regulation of E2F by pRB-family proteins[END_REF][START_REF] Stefano | E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes[END_REF]. The interaction of E2Fs with the pRB members tightly controls their transcriptional as well as their repressive functions on target-genes mostly involved in G 1 to S phase transition [START_REF] Harbour | The Rb/E2F pathway: expanding roles and emerging paradigms[END_REF]. If individual E2F proteins demonstrate overlapping functions in the control of cell cycle progression, they also display distinct specificities [START_REF] Degregori | Distinct roles for E2F proteins in cell growth control and apoptosis[END_REF]. Actually, only E2F1, E2F2 and E2F3 are able to induce apoptosis in fibroblasts [START_REF] Degregori | Distinct roles for E2F proteins in cell growth control and apoptosis[END_REF][START_REF] Johnson | Expression of transcription factor E2F1 induces quiescent cells to enter S phase[END_REF][START_REF] Qin | Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis[END_REF][START_REF] Wu | p53 and E2F-1 cooperate to mediate apoptosis[END_REF][START_REF] Kowalik | E2F1-specific induction of apoptosis and p53 accumulation, which is blocked by Mdm2[END_REF]. In addition, E2F1deficient mice exhibit a defect in thymocyte apoptosis as well as in negative selection demonstrating the role of E2F1 during the physiological apoptotic processes [START_REF] Field | E2F-1 functions in mice to promote apoptosis and suppress proliferation[END_REF][START_REF] Garcia | A role for E2F1 in the induction of apoptosis during thymic negative selection[END_REF].

Importantly, as these mice also develop tumors [START_REF] Field | E2F-1 functions in mice to promote apoptosis and suppress proliferation[END_REF][START_REF] Yamasaki | Tumor induction and tissue atrophy in mice lacking E2F-1[END_REF], it has been proposed that E2F1mediated apoptosis could contribute to its tumor suppressive function. E2F1-induced apoptosis occurs via both p53-dependent and -independent pathways, and involves the transcriptional activation of numerous pro-apoptotic E2F1-target genes including p14 ARF /p19 ARF , p73, apaf1 and caspases [START_REF] Wu | p53 and E2F-1 cooperate to mediate apoptosis[END_REF][START_REF] Hiebert | E2F-1:DP-1 induces p53 and overrides survival factors to trigger apoptosis[END_REF][START_REF] Hsieh | E2F1-induced apoptosis requires DNA binding but not transactivation and is inhibited by the retinoblastoma protein through direct interaction[END_REF]. Moreover, alternative models suggest that E2F1 also inhibits anti-apoptotic signals such as NF-kB, or sensitizes cells to pro-apoptotic stimuli (for review, see [START_REF] Ginsberg | E2F1 pathways to apoptosis[END_REF]). Recently, a link between E2F1 and the extrinsic apoptotic pathway also called death receptor-related apoptosis has been established in murine primary T cells [START_REF] Cao | The E2F-1 transcription factor promotes caspase-8 and bid expression, and enhances Fas signaling in T cells[END_REF] and post-mitotic neurons [START_REF] Hou | Activation of the Rb/E2F1 pathway by the nonproliferative p38 MAPK during Fas (APO1/CD95)-mediated neuronal apoptosis[END_REF]. FAS (also CD95, APO-1) is a 45-kDa cell surface protein which belongs to the tumor necrosis factor (TNF) / nerve growth factor (NGF) receptors family [START_REF] Itoh | The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis[END_REF]. Following stimulation by its cognate ligand (FasL) or by agonistic FAS antibodies, FAS oligomerizes and recruits several death-associated molecules, including the FADD adaptator (Fas-Associated Death Domain-containing protein, also MORT1) [START_REF] Boldin | A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain[END_REF][START_REF] Chinnaiyan | FADD, a novel death domaincontaining protein, interacts with the death domain of Fas and initiates apoptosis[END_REF] and the caspase-8 (also FLICE /MACH/Mch5) [START_REF] Boldin | Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1-and TNF receptor-induced cell death[END_REF][START_REF] Srinivasula | Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases[END_REF][START_REF] Muzio | FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex[END_REF], into a multimolecular signaling plateform named the Death-Inducing Signaling Complex (DISC; [START_REF] Kischkel | Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor[END_REF]). Further activation of caspase-8 by autoproteolytic cleavage at the DISC level initiates the apoptotic signaling cascade [START_REF] Medema | FLICE is activated by association with the CD95 death-inducing signaling complex (DISC)[END_REF][START_REF] Hirata | Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis[END_REF] which can either directly activate effector caspases or trigger the mitochondrial apoptosis loop amplification [START_REF] Scaffidi | Two CD95 (APO-1/Fas) signaling pathways[END_REF]. FAS-induced cell death is tightly controlled by proximal regulators, among which is c-FLIP (FLICE Inhibitory Protein), a procaspase-8-like protease deficient protein [START_REF] Goltsev | CASH, a novel caspase homologue with death effector domains[END_REF][START_REF] Han | MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclXL and initiates cell death[END_REF][START_REF] Hu | I-FLICE, a novel inhibitor of tumor necrosis factor receptor-1-and CD-95-induced apoptosis[END_REF][START_REF] Inohara | CLARP, a death effector domaincontaining protein interacts with caspase-8 and regulates apoptosis[END_REF][START_REF] Irmler | Inhibition of death receptor signals by cellular FLIP[END_REF][START_REF] Shu | Casper is a FADD-and caspase-related inducer of apoptosis[END_REF][START_REF] Srinivasula | FLAME-1, a novel FADD-like anti-apoptotic molecule that regulates Fas/TNFR1-induced apoptosis[END_REF]. c-FLIP is mainly expressed as a long (c-FLIP L ) and a short (c-FLIP S ) spliced isoforms, which can be both recruited to the DISC. Similar to caspase-8, c-FLIP L consists of two amino-terminal death effector domains (DED) followed by an unfunctional caspase homology domain [START_REF] Krueger | Cellular FLICEinhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex[END_REF]. To date, the role of c-FLIP L in apoptosis remains controversial. Indeed, pro-apoptotic, anti-apoptotic and proliferative functions have been reported [START_REF] Krueger | Cellular FLICEinhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex[END_REF][START_REF] Chang | c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis[END_REF][START_REF] Micheau | The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex[END_REF][START_REF] Boatright | Activation of caspases-8 and -10 by FLIP(L)[END_REF][START_REF] Mathas | c-FLIP mediates resistance of Hodgkin/Reed-Sternberg cells to death receptor-induced apoptosis[END_REF].

In contrast, only anti-apoptotic functions have been so far ascribed to c-FLIP S [START_REF] Krueger | Cellular FLICEinhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex[END_REF].

We had previously shown that E2F1 expression is faintly detectable in human lung adenocarcinomas [START_REF] Eymin | Distinct pattern of E2F1 expression in human lung tumours: E2F1 is upregulated in small cell lung carcinoma[END_REF]. As E2F1-deficient mice develop pulmonary adenocarcinoma with a high incidence [START_REF] Field | E2F-1 functions in mice to promote apoptosis and suppress proliferation[END_REF], we had postulated that low levels of E2F1 contribute to the carcinogenesis of human lung adenocarcinomas by creating a defective response of tumor cells to apoptotic stimuli. Consistent with such hypothesis, we demonstrate that E2F1 induces apoptosis in various human lung adenocarcinoma cell lines by a mechanism involving a specific downregulation of the c-FLIPs protein, leading to the activation of caspase-8 at the DISC level. Importantly, we also provide evidence that downregulation of c-FLIPs by E2F1 restores tumor cells sensitivity to death-receptor-mediated apoptosis as well as to lymphocytes T-mediated cytotoxicity, indicating that E2F1 could regulate the cellular response to the immune system.

Results

E2F1-induced apoptosis in lung adenocarcinoma cell lines requires caspase-8 activation and an intact E2F1 DNA-binding domain

We first tested the ability of E2F1 to trigger apoptosis in A549 and H1299 human lung adenocarcinoma cell lines using transient transfection with a vector encoding wild-type E2F1.

As compared to cells transfected with a control vector, enforced E2F1 expression strongly induced apoptosis in both cell lines (Figure 1a). In order to confirm the results of transient transfections, we then established stable E2F1 or E2F1(E132) DNA-binding defective mutant inducible clones in the H358 cell line derived from a human lung adenocarcinoma, using the doxycyclin-inducible expression system (Tet-On). Of note, E2F1 expression was undetectable in H358 cells (Figure 1b, upper panel). Several stable transfectant clones were obtained and were found to give similar results. Therefore, data presented with H358/Tet-On/E2F1 clone 6 and H358/Tet-On/E2F1(E132) clone 22 are representative of all clones tested. As shown in figure 1b (upper panel), expression of E2F1 and E2F1(E132) proteins was strongly induced after 48 hours of doxycyclin treatment and persisted even after 6 days of continuous induction. In H358/Tet-On/E2F1 cells, apoptosis was clearly detected after 72 hours of doxycyclin treatment and reached 20% of total cells after 6 days (Figure 1b, lower panel). In contrast, and consistent with previous studies [START_REF] Hsieh | E2F1-induced apoptosis requires DNA binding but not transactivation and is inhibited by the retinoblastoma protein through direct interaction[END_REF][START_REF] Phillips | Induction of DNA synthesis and apoptosis are separable functions of E2F-1[END_REF], apoptosis was never observed in H358/Tet-On/E2F1(E132) cells (Figure 1b, lower panel). Appearance of apoptotic cells upon E2F1 expression correlated with the cleavage of procaspases-8 and -3 in their p43/41 and p17 active fragments respectively, followed by the activation of procaspases-6 and -7 (Figure 1c).

Interestingly and consistent with previous data [START_REF] Nahle | Direct coupling of the cell cycle and cell death machinery by E2F[END_REF], E2F1 also increased procaspase-7 expression level. Of note, activation of this proteolytic cascade was never detected following E2F1(E132) overexpression (data not shown). In order to confirm the involvement of the caspase-8 initiator and the caspase-3 effector during E2F1-mediated apoptosis, we finally tested the effect of two specific caspases-8 and -3 inhibitors, namely z-IETD-fmk and z-VED-fmk respectively. As shown in Figure 1d, both inhibitors significantly reversed the ability of E2F1 to induce apoptosis. In contrast, a specific caspase-9 inhibitor (z-LEDH-fmk) had no significant effect and the cytochrome c was never released from mitochondria in response to E2F1 expression suggesting that the mitochondrial amplification loop was not involved in our model (data not shown). Overall, these results indicate that E2F1 requires an intact DNA-binding domain and caspases-8 and -3 activities to trigger apoptosis in adenocarcinoma cell lines.

E2F1 activates caspase-8 at the DISC level

Until now, two distinct cellular models of FAS signaling pathway activation have been described. In type I cells, the death signal is initiated by the accumulation of activated caspase-8 at the DISC level. In type II cells, procaspase-8 expression level is low and the caspase cascade requires further amplification through the mitochondrial loop [START_REF] Scaffidi | Two CD95 (APO-1/Fas) signaling pathways[END_REF]. The inhibition of E2F1-dependent apoptosis by the z-IETD-fmk caspase-8 inhibitor prompted us to analyze the DISC components. We first observed that E2F1 did not affect FAS, FasL or FADD protein expression, nor increased the amount of the FAS receptor at the membrane (data not shown). Caspase-8 activation at the DISC results mainly from the binding of FasL to the FAS receptor but can also occur in the absence of FasL triggering [START_REF] Fulda | Betulinic acid triggers CD95 (APO-1/Fas)-and p53-independent apoptosis via activation of caspases in neuroectodermal tumors[END_REF][START_REF] Aragane | Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L[END_REF][START_REF] Cock | The anti-cancer drug etoposide can induce caspase-8 processing and apoptosis in the absence of CD95 receptor-ligand interaction[END_REF][START_REF] Micheau | Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs[END_REF]. To determine whether E2F1-induced cell death involved an interaction between FAS and FasL, we used the antagonistic ZB4 anti-Fas antibody which inhibits FAS/FasL binding. Our data showed that treating cells with ZB4, at a concentration that totally prevented CH11-mediated cytotoxicity, did not modulate the ability of E2F1 to induce apoptosis as detected by Hoechst staining (Figure 2a, left panel) or caspase-3 activation (Figure 2a, right panel). These results indicated that a FAS/FasL interaction did not play a central role in E2F1-induced apoptosis. In keeping with these data, we did not detect any FasL secretion into the culture medium, nor a modulation of FasL expression on the membrane in response to E2F1 (data not shown). In order to analyze the DISC, FAS co-immunoprecipitation experiments were performed in H358/Tet-On/E2F1 clones. E2F1 induction led to the recruitment of FADD, caspase-8 and caspase-10 at the FAS DISC (Figure 2b). Importantly, the procaspase-8 was activated as reflected by the appearance of its p43/41 active fragments in FAS co-immunoprecipitates (Figure 2b). In contrast, and consistent with the FAS/FasL interaction being not a critical determinant of E2F1-mediated apoptosis (Figure 2a), we did not observe any recruitment of FasL to the FAS receptor in response to E2F1. Moreover, neither FADD, nor activated caspase-8 were co-immunoprecipitated at the FAS DISC in E2F1(E132)-expressing cells (data not shown) which was in agreement with the absence of apoptosis. Finally, as DISC formation and caspase-8 activation stimulate the clustering of the FAS receptor at the cell surface (47), we used confocal laser scanning microscopy to further analyze the FAS receptor subcellular distribution in our cells. Exposure of 293FasL cells to agonistic anti-FAS CH11 mAb which induces FAS clustering, was used as a positive control (Figure 2c, left panel). As expected, E2F1 induced the clustering of FAS whereas E2F1(E132) did not (Figure 2c).

Overall, these results demonstrate that E2F1 activates the DISC. Importantly, these effects do not require FAS/FasL interaction.

E2F1 downregulates c-FLIPS protein expression to induce apoptosis

As our results indicated that E2F1 triggered cell death by affecting downstream effectors of the FAS signaling pathway, we then focused on the proximal regulators of procaspase-8 activation at the DISC level. c-FLIP (cellular FLICE-inhibitory protein) is a specific endogenous caspase-8 inhibitor [START_REF] Irmler | Inhibition of death receptor signals by cellular FLIP[END_REF]. Both c-FLIP long (c-FLIP L ) and c-FLIP short (c-FLIP S ) isoforms can be incorporated into the DISC and inhibit specific steps of caspase-8 activation [START_REF] Krueger | Cellular FLICEinhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex[END_REF]. Consequently, elevated levels of c-FLIP protein protect cells from death receptor-mediated apoptosis. Interestingly, E2F1 but not E2F1(E132) induction was associated with a modest increase and a strong decrease of c-FLIP L and c-FLIPs protein levels respectively (Figure 3a). These effects correlated with the appearance of apoptosis 72 hours post-induction and persisted even after 6 days of doxycyclin treatment. Of note, E2F1 expression also stimulated the cleavage of c-FLIP L into its p43 fragment. In the context where E2F1-induced apoptosis requires the specific downregulation of c-FLIP S as suggested by our results, we postulated that ectopic expression of c-FLIP S could block E2F1-mediated programmed cell death. To test this hypothesis, H358/Tet-On/E2F1 cells were stably infected with a VSV-FLIPshort (FLIP S ) expression vector or a control mock vector. As compared to mock-infected cells, c-FLIP S -overexpressing cells were significantly more resistant to apoptosis induced by E2F1 (Figure 3b). In contrast, silencing of c-FLIPs using RNA interference in H358/Tet-On/E2F1 cells enabled a 3-fold increase in the percentage of apoptotic cells upon E2F1 expression (Figure 3c). Interestingly, this was detected after 48 hours of E2F1 induction. Taken together, these results demonstrate that c-FLIP S is a potent inhibitor of E2F1-induced apoptosis. Importantly, c-FLIPS protein expression was also specifically increased in E2F1-deficient MEFs as compared to wild-type MEFs while c-FLIP L status was not affected (Figure 3d). These results indicate that c-FLIP S is also an E2F1-target in primary cells.

Downregulation of c-flip s mRNA by E2F1 requires protein synthesis

We further investigated the molecular mechanisms involved in E2F1-mediated c-FLIP S downregulation. To this end, we used RT-PCR to analyze the effect of E2F1 on c-flip l and c-flip s mRNA levels. As shown in figure 4a, expression of E2F1 induced a specific decrease in c-flip s mRNA level which was detected upon 72 hours of doxycyclin treatment, whereas c-flip l mRNA level remained unaffected. In the same conditions, E2F1(E132) expression did not affect mRNA level of both c-flip s and c-flip l , confirming the requirement of the E2F1 DNA-binding domain to regulate c-FLIPs. Importantly, E2F1 was not able anymore to downregulate c-flip s mRNA level in the presence of cycloheximide, a protein synthesis inhibitor, indicating that de-novo protein synthesis is required for E2F1 to regulate c-flip s (Figure 4b).

E2F1 sensitizes NSCLC cells to death receptor mediated apoptosis

Our data demonstrated that E2F1 overexpression was sufficient to activate the FAS signaling pathway by affecting c-FLIP S expression, even in the absence of death receptor ligands. As the role of endogenous c-FLIP proteins, and more specifically of c-FLIPS, in resistance to FAS-related apoptosis had been established [START_REF] Irmler | Inhibition of death receptor signals by cellular FLIP[END_REF], we tested whether E2F1 could affect the response of lung adenocarcinoma cells to death receptor stimuli. Using transient transfections, we first demonstrated that E2F1 overexpression sensitized A549 and H1299 cells to apoptosis induced by the agonistic FAS CH11mAb as compared to control-transfected cells (Figure 5a). Same results were observed in H358/Tet-On/E2F1 clones which became sensitive to CH11 treatment in the presence of E2F1 (Figure 5b, upper). In contrast, E2F1(E132)-expressing cells remained highly resistant (Figure 5b, upper panel). Furthermore, E2F1-expressing cells were also more sensitive to FasL and TRAIL treatments as compared to uninduced or E2F1(E132)-expressing cells (Figure 5b, lower panel). Interestingly, E2F1 did not increase the cytotoxic effects of etoposide or paclitaxel (Figure 5c) indicating that E2F1 specifically sensitized H358 cells to death receptor stimuli. Importantly, these effects were not restricted to lung adenocarcinoma cell lines. Indeed, we demonstrated that E2F1-/-MEF were more resistant to FasL or TRAIL treatment than wild-type MEF (Figure 5d), although they expressed the same level of FAS, DR4 and DR5 at the membrane (data not shown). These results support the idea that endogenous E2F1 affects the FAS death pathway downstream of the receptor level. Finally, we asked whether downregulation of c-FLIP S was sufficient to restore tumor cells sensitivity to apoptosis triggered by death receptors stimuli.

As shown in Figure 5e, neutralisation of c-flip s using siRNA highly sensitizes H358/Tet-On/E2F1 clones to CH11 or TRAIL treatment when compared to cells transfected with mismatch siRNA. Interestingly, concomittant E2F1 induction in the same cells did not significantly potentiate the effect of c-flip s neutralization, suggesting that E2F1 sensitizes these cells to death receptor mediated apoptosis through c-FLIP S downregulation.

E2F1 enhances tumor cell sensitivity to lymphocytes T-mediated cytotoxicity

FAS/FasL interactions and perforin/granzyme B pathways are the two main lytic mechanims used by cytotoxic T cells [START_REF] Kagi | Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity[END_REF][START_REF] Lowin | Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways[END_REF]. Recent data provided evidence that an immune response might occur in NSCLCs and especially that effector T cells might contribute to tumor regression [START_REF] Mami-Chouaib | Antitumor cytotoxic T-lymphocyte response in human lung carcinoma: identification of a tumorassociated antigen[END_REF]. In addition, it was demonstrated that c-FLIP protein expression enabled tumor cells to escape from T cell immunity "in vivo" [START_REF] Djerbi | The inhibitor of death receptor signaling, FLICE-inhibitory protein defines a new class of tumor progression factors[END_REF][START_REF] Medema | Immune escape of tumors in vivo by expression of cellular FLICE-inhibitory protein[END_REF]. Since we established that E2F1 regulated the cellular response to death receptor stimuli by downregulating c-FLIP S , we next tested its ability to sensitize H358 cancer cells to cytolytic T cells. To do so, a lymphocytes T-mediated cytotoxic assay was set up in the presence of EGTA which blocks the perforin pathway. In this context, the cellular lysis is strictly dependent on a functional FAS/FasL interaction. The Jurkat T cells expressing high levels of the FAS receptor were used as positive control of effective lysis by activated T lymphocytes (Figure 6). When H358/Tet-On/E2F1 cells cultured for 72 hours in the presence or in the absence of doxycyclin were used as targets, the lysis mediated by cytotoxic T cells was significantly increased in E2F1-expressing cells as compared to uninduced cells (Figure 6). Therefore, we concluded from these data that E2F1 also enhances the response of tumor cells to the lymphocytes Tdependent cytotoxicity.

c-FLIPS is overexpressed in human lung adenocarcinomas

Finally, we investigated whether the c-FLIP S isoform is deregulated in human lung adenocarcinomas which display undetectable E2F1 protein expression [START_REF] Eymin | Distinct pattern of E2F1 expression in human lung tumours: E2F1 is upregulated in small cell lung carcinoma[END_REF]. To this end, we analyzed E2F1 and c-FLIP protein expression by western blotting on 12 tumor samples and 5 matched normal lung tissues. Consistent with our previous study [START_REF] Eymin | Distinct pattern of E2F1 expression in human lung tumours: E2F1 is upregulated in small cell lung carcinoma[END_REF], E2F1 protein expression was hardly detectable in the tumor samples (Figure 7). Interestingly, c-FLIP S protein expression was strongly enhanced in these samples as compared to normal lung tissues whereas c-FLIP L protein expression was not altered (Figure 7). Overall, these results show that c-FLIP S is specifically overexpressed in lung adenocarcinomas. Moreover, they provide evidence that both c-FLIP protein isoforms can be differentially expressed.

Discussion

A large body of evidence has established that apoptosis can occur through both intrinsic (caspase-9 mediated) and extrinsic (caspase-8 mediated) death pathways. The ability of E2F1 to promote apoptosis by activating the intrinsic pathway has been well documented, notably through transcriptional activation of apaf-1 or caspase-9 genes (for review, see [START_REF] Ginsberg | E2F1 pathways to apoptosis[END_REF]).

In contrast, the relationships between E2F1 and the extrinsic pathway have been less studied.

Here, we demonstrate that E2F1 triggers apoptosis in various human lung adenocarcinoma cell lines by a mechanism involving the transcriptional inhibition of c-flip s thus leading to the activation of caspase-8 at the DISC level. Importantly, we find that c-FLIP S protein is specifically overexpressed in lung adenocarcinoma which display a low level of E2F1 expression, and provide evidence that E2F1 restores tumor cells sensitivity to death-receptor stimuli, by downregulating c-FLIP S expression. Overall, our data identify a new mechanistic link between E2F1 and the death-receptor signaling pathway and suggest that downregulation of E2F1 expression level contributes to the immune escape of lung adenocarcinoma tumor cells.

E2F1 can induce apoptosis via several p53-dependent or -independent mechanisms, either through direct transactivation of proapoptotic genes such as p73 [START_REF] Irwin | Role for the p53 homologue p73 in E2F-1-induced apoptosis[END_REF][START_REF] Stiewe | Role of the p53-homologue p73 in E2F1-induced apoptosis[END_REF], apaf-1 (apoptosis protease-activating factor 1) [START_REF] Moroni | Apaf-1 is a transcriptional target for E2F and p53[END_REF][START_REF] Furukawa | Apaf-1 is a mediator of E2F-1-induced apoptosis[END_REF] and caspases [START_REF] Nahle | Direct coupling of the cell cycle and cell death machinery by E2F[END_REF]or transcriptional repression [START_REF] Hsieh | E2F1-induced apoptosis requires DNA binding but not transactivation and is inhibited by the retinoblastoma protein through direct interaction[END_REF][START_REF] Phillips | Induction of DNA synthesis and apoptosis are separable functions of E2F-1[END_REF]. Here, we show that E2F1 is able to induce a p53-independent apoptosis through the downregulation of the c-FLIP S anti-apoptotic protein. So far, a few studies have reported a link between E2F1 and the death-receptor signaling machinery [START_REF] Cao | The E2F-1 transcription factor promotes caspase-8 and bid expression, and enhances Fas signaling in T cells[END_REF][START_REF] Hou | Activation of the Rb/E2F1 pathway by the nonproliferative p38 MAPK during Fas (APO1/CD95)-mediated neuronal apoptosis[END_REF][START_REF] Phillips | E2F-1 potentiates cell death by blocking antiapoptotic signaling pathways[END_REF][START_REF] Wang | Regulation of Rb and E2F by signal transduction cascades: divergent effects of JNK1 and p38 kinases[END_REF][START_REF] Bi | Cleavage of E2F-1-regulating proteins and activation of E2F-1 during CD95-induced death of thymocytes[END_REF]. In this work, we found that E2F1 activates the proteolytic cleavage of caspase-8 at the DISC and induces the clustering of the FAS receptor in the absence of FasL binding. Such model in which death receptors are activated without the need of their respective death ligand has been previously reported in a context of UVB exposure [START_REF] Aragane | Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L[END_REF]. Moreover, we showed that caspase-8 activation by E2F1 is specifically associated with the downregulation of c-FLIP S . Originally, c-FLIP proteins were solely considered as inhibitors of death receptor-induced apoptosis, functioning as procaspase-8 competitors for binding to FADD at the activated DISC [START_REF] Tschopp | Inhibition of fas death signals by FLIPs[END_REF][START_REF] Jiang | Induction of tumor suppression and glandular differentiation of A549 lung carcinoma cells by dominant-negative IGF-I receptor[END_REF][START_REF] Fulda | Metabolic inhibitors sensitize for CD95 (APO-1/Fas)induced apoptosis by down-regulating Fas-associated death domain-like interleukin 1converting enzyme inhibitory protein expression[END_REF]. In this context, c-FLIP S appeared to be more efficient than c-FLIP L [START_REF] Inohara | CLARP, a death effector domaincontaining protein interacts with caspase-8 and regulates apoptosis[END_REF][START_REF] Shu | Casper is a FADD-and caspase-related inducer of apoptosis[END_REF][START_REF] Thome | Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors[END_REF][START_REF] Bin | The short splice form of Casper/c-FLIP is a major cellular inhibitor of TRAIL-induced apoptosis[END_REF][START_REF] Kirchhoff | TCR-mediated upregulation of c-FLIPshort correlates with resistance toward CD95-mediated apoptosis by blocking death-inducing signaling complex activity[END_REF]. Interestingly, recent works showed that recruitement of c-FLIP L to the DISC could also promote caspase-8 activation [START_REF] Chang | c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis[END_REF][START_REF] Micheau | The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex[END_REF][START_REF] Boatright | Activation of caspases-8 and -10 by FLIP(L)[END_REF]. In our cells, we never detected the presence of c-FLIP L at the DISC level. Thus, it is tempting to speculate that E2F1, by altering the stoechiometry of c-FLIP S and c-FLIP L molecules, enables activation of caspase-8 at the DISC and triggers programmed cell death. However, as c-FLIP S neutralization does not induce apoptosis on its own in our cells, E2F1 probably activates other collateral signals which collaborate with the downregulation of c-FLIP S to trigger caspase-8 activation and cell death. Interestingly, it was recently shown that loss of Rb-E2F repression resulted in caspase-8 mediated apoptosis through inactivation of focal adhesion kinase [START_REF] Lieman | Loss of Rb-E2F repression results in caspase-8 mediated apoptosis through inactivation of focal adhesion kinase[END_REF].

Both c-FLIP isoforms arise from alternative splicing and until now, the molecular mechanisms controlling their relative expression remain largely unknown. In this study, we demonstrated that E2F1 differentially affects the expression of c-FLIP S and c-FLIP L proteins in human cell lines. Furthermore, we also showed the existence of a distinct expression pattern of the two isoforms in a few samples of human lung adenocarcinoma. Altogether, these results are consistent with the notion that c-FLIP S and c-FLIP L are differentially regulated and exhibit distinct functions. Recent data demonstrated that the level of cellular FLIP is regulated both at transcriptional and post-traductional levels, the latter involving the ubiquitin-proteasome pathway [START_REF] Fukazawa | Accelerated degradation of cellular FLIP protein through the ubiquitin-proteasome pathway in p53-mediated apoptosis of human cancer cells[END_REF][START_REF] Kim | An inducible pathway for degradation of FLIP protein sensitizes tumor cells to TRAIL-induced apoptosis[END_REF]. We showed that E2F1 induces a specific decrease of c-flip s mRNA level by a mechanism requiring its DNA-binding domain. Interestingly, a recent work demonstrated the ability of c-myc to bind and repress the human FLIP promoter [START_REF] Ricci | Direct repression of FLIP expression by c-myc is a major determinant of TRAIL sensitivity[END_REF]. Thus, E2F1 could act in the same way to downregulate c-flip s mRNA. Inhibition of NF-kB activity is one of the molecular mechanism by which E2F1 promotes apoptosis [START_REF] Phillips | E2F-1 potentiates cell death by blocking antiapoptotic signaling pathways[END_REF][START_REF] Tanaka | E2F1 and c-Myc potentiate apoptosis through inhibition of NF-kappaB activity that facilitates MnSODmediated ROS elimination[END_REF].

As c-flip is a direct NF-kB-target gene [START_REF] Micheau | NF-kappaB signals induce the expression of c-FLIP[END_REF], E2F1 could also affect c-flip s transcription through NF-kB inhibition. Although these two hypotheses do not fit well with the observation that E2F1 only affects c-flip s mRNA level and that "de novo" protein synthesis is required, it remains possible that E2F1 binds the FLIP promoter and modulates the recruitement of specific co-factors, in order to differentially affect the transcription of both flip isoforms.

Alternatively, E2F1 could specifically downregulate the stability of c-flip s mRNA or modulate the expression level of proteins controlling the alternative splicing of both isoforms.

We are currently testing these hypotheses. Of note, we observed that pre-treatment of cells with proteasome inhibitors did not prevent the ability of E2F1 to downregulate c-FLIP S (data not shown), indicating that additional c-FLIP S control at the protein level is not involved in this process.

We provided evidence that E2F1 sensitized primary and tumor cells to FasL-or TRAIL-mediated apoptosis. Such enhanced resistance to Fas-triggered apoptosis has been already described in E2F1-/-cerebellar granule neurons [START_REF] Hou | Activation of the Rb/E2F1 pathway by the nonproliferative p38 MAPK during Fas (APO1/CD95)-mediated neuronal apoptosis[END_REF], thymocytes (59) and lymphocytes [START_REF] Cao | The E2F-1 transcription factor promotes caspase-8 and bid expression, and enhances Fas signaling in T cells[END_REF]. Altogether, these data are consistent with E2F1 being a critical determinant of the cellular response to death-receptor activation, at least in some cellular types. In addition, this is to our knowledge the first study demonstrating the involvement of E2F1 in the cellular response to TRAIL. High c-FLIP expression has been found in many tumor cells and was correlated with resistance to CD95-and TRAIL-induced apoptosis [START_REF] Tschopp | Inhibition of fas death signals by FLIPs[END_REF]. Recently, a genetic screen identified that the c-FLIP S isoform confered resistance to TRAIL in sensitive cells [START_REF] Burns | Identification of inhibitors of TRAIL-induced death (ITIDs) in the TRAIL-sensitive colon carcinoma cell line SW480 using a genetic approach[END_REF]. Furthermore, c-FLIP S was reported to be more efficient than c-FLIP L in the inhibition of CD95-mediated apoptosis in T cells [START_REF] Schmitz | Resistance of short term activated T cells to CD95-mediated apoptosis correlates with de novo protein synthesis of c-FLIP(short)[END_REF]. Interestingly, E2F1-deficient mice accumulate mature T cells due to a defect in thymocyte apoptosis and negative selection [START_REF] Garcia | A role for E2F1 in the induction of apoptosis during thymic negative selection[END_REF][START_REF] Yamasaki | Tumor induction and tissue atrophy in mice lacking E2F-1[END_REF], a process functioning mainly through death-promoting ligands. Whether c-FLIP S is overexpressed in E2F1-/-thymocytes remains to be determined. In our model, selective neutralization of c-FLIP S by siRNA was sufficient to restore tumor cells sensitivity to both Fas-and TRAIL-mediated apoptosis and concomittant induction of E2F1 in these cells did not lead to an additive effect. Therefore, these data are in favor of E2F1 sensitizing these tumor cells to death-receptor mediated apoptosis mostly through c-FLIP S downregulation. In this setting, sensitization is likely to be mediated by a facilitated caspase-8 activation in the absence of c-FLIP S . The death receptor pathway represents one of the mechanisms by which immune cells kill target tumor cells. In this context, we also provided evidence that E2F1 enhanced tumor cell sensitivity to lymphocytes T-mediated cytotoxicity. Interestingly, c-FLIP expression was associated with tumor escape from T-cell immunity and enhanced tumor progression in "in vivo" experimental studies pointing to a role of FLIP as a tumor progression factor [START_REF] Djerbi | The inhibitor of death receptor signaling, FLICE-inhibitory protein defines a new class of tumor progression factors[END_REF][START_REF] Medema | Immune escape of tumors in vivo by expression of cellular FLICE-inhibitory protein[END_REF][START_REF] French | Defective death receptor signaling as a cause of tumor immune escape[END_REF].

Many tumors are resistant to Fas-and TRAIL-induced cell death. Production of decoy receptors [START_REF] Roth | Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis[END_REF], expression of FasL to perform the so-called tumor counterattack [START_REF] Hahne | Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape[END_REF], and upregulation of apoptosis inhibitors expression [START_REF] French | Defective death receptor signaling as a cause of tumor immune escape[END_REF][START_REF] Mitsiades | Activation of NF-kappaB and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: therapeutic implications[END_REF] are among the molecular mechanisms involved in this process. We previously reported the frequent decrease of FAS expression in NSCLC [START_REF] Viard-Leveugle | Frequent loss of Fas expression and function in human lung tumours with overexpression of FasL in small cell lung carcinoma[END_REF]. Importantly, it has been shown than escape from T cell-mediated immunity "in vivo" by c-FLIP overexpression was not limited to tumors with high FAS surface expression [START_REF] Medema | Immune escape of tumors in vivo by expression of cellular FLICE-inhibitory protein[END_REF]. In the light of these data, the low level of E2F1 protein expression in NSCLC with reduced FAS expression could be another way to block FAS-triggered cell death, therefore contributing to the immune escape of tumor cells. In this setting, E2F1 restoration might represent a key step in therapeutic strategies to reconstitute the ability of tumor cells to undergo apoptosis.

Materials and methods

Cell lines, expression vectors and transfection

H358, A549 and H1299 human lung carcinoma cell lines as well as Jurkat T cells were cultured in RPMI-1640 medium (GibcoBRL, Life Technologies, Cergy Pontoise, France), supplemented with 10% (v/v) heat-inactivated fetal calf serum (GibcoBRL), 2mM Lglutamine and 100U/ml penicillin/streptomycin, in 5% CO 2 at 37°C. Primary murine embryonic fibroblasts (MEF) derived from wild-type and E2F1 -/-mice (kindly gift from Dr.

Nevins) and 293-FasL embryonic kidney cells stably expressing human Fas ligand were maintained in DMEM (GibcoBRL) supplemented with 10% (v/v) heat-inactivated fetal calf serum and 2mM L-glutamine. To ensure exponential growth, cells were resuspended in fresh medium 24 hours before transfection or treatment. Stable inducible E2F1 (wild-type) or E2F1(132) (DNA-binding defective mutant) clones derived from H358 cells were obtained using a modified tetracyclin-regulated inducible expression system (Tet-On System, Clontech, Ozyme, Saint Quentin en Yvelines, France) as previously described [START_REF] Eymin | p14ARF induces G2 arrest and apoptosis independently of p53 leading to regression of tumours established in nude mice[END_REF]. E2F1 expression was induced upon 1µg/ml doxycyclin treatment. Inducible clones stably infected with either VSV-FLIP S expression vector or with a mock vector were provided by O. Micheau (INSERM U517, Dijon, France). Transient transfections of A549 and H1299 cells with either control pcDNA3.1/GFP or pcDNA3.1/E2F1 expression vectors were carried out using JetPEI™ (Polyplus Transfection, Illkirch, France) according to the manufacturer's protocol. In some experiments, geniticin (800 µg/ml) was added to the culture medium 48 hours after transfection and selection was performed during 3 additional days before analysis.

Tissue samples

Tissue samples taken at surgical resection of lung tumors were immediately frozen and kept at -80°C until use. They consisted of 12 adenocarcinoma according to the 1999 WHO international histological classification of lung tumors and 5 matched normal lung samples.

Reagents and antibodies

Agonistic anti-human Fas monoclonal antibody (clone CH-11) and caspase inhibitors z-IETD-fmk, z-DEVD-fmk and Z-LEDH-fmk were obtained from MBL (MBL, France Biochem, Meudon). For each experiment, caspase inhibitors (2 µM) were added to the culture medium 2 hours prior addition of doxycyclin (1µg/ml). Treatment with CH-11 mAb (250ng/ml) was carried out for 48 hours. Soluble Fas ligand was collected from 293-FasL supernatant. Recombinant soluble human FLAG-tagged FasL, Flag-tagged TRAIL and their enhancer were purchased from Alexis (San Diego, CA, USA). Etoposide (VP16), paclitaxel (Taxol™) and cycloheximide were obtained from Sigma (Saint Quentin Fallavier, France).

Cycloheximide was used at 10µg/ml for 12 hours.

Monoclonal antibodies used included anti-procaspase-8 (5F7), anti-procaspase-10 (4C1) and anti-Fas (ZB4) (MBL), anti-FADD (1F7), anti-FasL (G247-4), anti-procaspase-6 and anti-E2F1(KH95) (Pharmingen BD, San Diego, CA, USA), anti-HSP 70 (Affinity Bioreagents, Golden, CO, USA), anti-actin (Santa Cruz, CA, USA), anti-E2F1(Ab4; Neomarkers, CA, USA), anti-Fas (APO-1 and 3D5) and anti-FLIP (NF6) (Alexis). To block FasL/Fas interaction, anti-Fas (ZB4) was used at 2 µg/ml. Hamster anti-mouse Fas (Jo2) and hamster IgG2 control were purchased from Pharmingen BD. Polyclonal antibodies used included anti-procaspase-3 and anti-active caspase 3 (Pharmingen BD), anti-procaspase-7 (Cell Signaling, Beverly, MA, USA), anti-procaspase-10 (Sigma), anti-E2F1 (Santa Cruz Biotechnology) and anti-FLIP (Santa Cruz Biotechnology).

Cell viability, apoptotic and cytotoxic assays

Apoptosis was evaluated by scoring the percentage of apoptotic cells on 500 cells after Hoescht 33342 staining. Cytotoxic experiments were performed using the methylene blue colorimetric assay. Briefly, stable inducible clones were seeded at 10 4 cells/well into a 96wells plate and were co-treated for 96 hours with increasing concentrations of cytotoxic drugs in the presence or absence of doxycyclin. Cells were then fixed in 100% ethanol, stained with 1% methylene blue in 0.01M borate buffer pH 8.5 for 15 min and optical density was measured at 630nm after elution in 0.1N HCl. Absorbance values were expressed as a percentage of untreated controls. Allogenic T cells cytotoxic assays against tumor target cells were measured in standard 24 hours 51 Cr-release assays as previously described [START_REF] Plumas | Tumor B cells from non-Hodgkin's lymphoma are resistant to CD95 (Fas/Apo-1)-mediated apoptosis[END_REF]. Briefly, 104 51 Cr-labeled target cells were mixed with the effector cells at different E/T ratios (25/1 to1/1) with E=effector cells and T=target cells. The radioactivity in supernatants was counted after 24 hours incubation at 37°C in 5% CO2. The percentage of specific lysis was calculated according to the following formula :% lysis = 100X (ER -SR)/(MR -SR), where ER, SR and MR represent experimental, spontaneous, and maximum 51 Cr-release, respectively. All expressed values were derived from average quadruplicate determinations. Effector allogenic T cells were obtained from unidirectional mixed lymphocyte reaction (MLR) as previously described. To induce FasL expression at the cell surface, effector cells were preincubated for 2 hours with a mixture of 5ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1.5µg/ml Ca2+ ionophore ionomycin (Sigma). Cytotoxic tests were performed on E2F1 stable inducible clones which were pretreated or not with doxycyclin for 72 hours, then used as target cells in complete medium supplemented with 15% human A serum in the presence or absence of 3mM EGTA/4mM Mg 2+ . Jurkat T cells treated in the same conditions were used as positive control target cells.

Western Blot and immunoprecipitation analyses

For immunoblotting, cells washed three times in PBS or frozen tissue samples were lysed in RIPA buffer (150mM NaCl, 50mM Tris HCl pH 8, 0.1% SDS, 1% Nonidet P40, 0.5% Na deoxycholate, 0.1mM PMSF, 2.5µg/ml pepstatin, 10µg/ml aprotinin, 5µg/ml leupeptin, 0.2mM Na 3 VO 4 ) for 30 min on ice and pelleted. Protein concentration was determined using the Biorad Dc protein assay. Proteins (40µg-80µg) were then separated in 10-12% SDS-PAGE gels and electroblotted onto PVDF membranes. Membranes were incubated overnight at +4°C with primary antibodies and proteins were detected using horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, USA). After washing, the blots were revealed using the ECL chemiluminescence method (Amersham, Les Ulis, France), according to the manufacturer's protocol.

For DISC immunoprecipitation, 50x10 6 cells were lysed in lysis buffer (20mM Tris-HCl pH 7.4, 150mM NaCl, 1% NP40, 2mM sodium vanadate, 1mM AEBSF and 10µg/ml aprotinin) for 15 min on ice and supernatants were collected by centrifugation at 12000 rpm for 15 min. Supernatants were precleared with Sepharose 6B (Sigma) for 1 hour at +4°C and immunoprecipitation of the FAS receptor was further performed overnight at +4°C on precleared lysates in presence of protein G sepharose and 2µg of FLAG-tagged-Fc-FasL (provided by Olivier Micheau). Protein G sepharose beads were spun down, washed 4 times with lysis buffer, resuspended in SDS gel sample buffer and boiled at 95°C for 4 min.

Immunoprecipitates were separated in 12% SDS/PAGE gel and immunoblotted with either anti-Fas (C-20, Santa-Cruz), anti-FADD (1F7, mouse IgG1, Immunotech), anti-caspase-8 (5F7, mouse IgG2b, Immunotech) or anti-caspase-10 (4C1, mouse IgG1, MBL) antibodies.

Secondary antibodies used were anti-mouse HRP-IgG1 (Southern Biotechnology) to detect FADD and caspase-10, anti-mouse HRP-IgG2b (Southern Biotechnology) to detect caspase-8 or anti-rabbit HRP to detect Fas (C-20).

Indirect immunofluorescence and Fas clustering studies

Transfected A549 and H1299 cells were fixed in -20°C acetone for 5 min. After extended washes, non specific binding sites were saturated for 45 min at RT in 2% BSA, 10% normal goat serum, 1X PBS and incubation with anti-E2F1 mAb (Ab4, 1/2000) was carried out for 2 hours at RT in PBS, 2% BSA. After three PBS washes, Alexa TM 568 goat antimouse IgG (H+L) conjugate (2mg/ml, 1/1000, Interchim Montluçon, France) was added and cells were further incubated for 30 min in dark at 37°C. Cells were then washed three times in PBS, counterstained with Hoescht 33342 and observed using an Olympus microscope. For Fas clustering analysis, 10 4 H358 stable inducible cells were seeded onto immunofluorescence slides and cultured in the presence or in the absence of doxycyclin for 6 days. 293FasL cells treated for 18 hours with 250ng/ml anti-Fas agonistic CH11 mAb were used as a positive control. Cells were then fixed in 3% paraformaldehyde/PBS for 10 min, washed twice with PBS for 10 min, preincubated with 1% BSA for 15 min, and incubated for 2 hours at RT with anti-Fas mAb (Zb4) in 1% BSA, PBS. Samples were washed twice in PBS and stained using the Alexa TM 568 goat anti-mouse IgG(H+L) conjugate. Cells were visualized using a confocal laser (Spectra Physics) microscope (Zeiss, LSM510). For all experiments, a nonrelevant isotype matching antibody was used as a negative control (data not shown).

Transfection of siRNA oligonucleotides

The siRNA oligonucleotides were synthesized by Eurogentec and resuspended according to the manufactuter's protocol (Eurogentec, Seraing, Belgium). The sequence designed to target specifically human flip short mRNA was as follow: 5-CACCCUAUGCCCAUUGUCCTT-3'. The scrambled siRNA oligonucleotide used as control for all RNA interference experiments was as follow: 5-ACAUACGCUUUCGCCCCUTT-3'.

For siRNA transfection, stable E2F1-inducibles clones were seeded at 5x10 5 cells/well into a 6-well plate. On the next day, the cells were transfected with siRNA oligonucleotides duplex using Oligofectamine™ (Invitrogen, Cergy Pontoise, France) according to the manufacturer's instructions. Briefly, 10µl of siRNA duplex (at 20µM) were mixed with 185µl of serum-free RPMI medium. 3µl of Oligofectamine ™ was added to 12µl of RPMI serum-free medium, vortexed, incubated at RT for 10 min and gently mixed with the siRNA duplex solution.

Mixture was incubated for 20 min at RT and added to each well. 10% fetal calf serum was added 4 hours after transfection. Doxycyclin was added or not in culture medium 24 hours after transfection and cells were cultured additionally for 48 hours before analysis. In some experiments, cells were transfected for 42 hours, then treated for 4 hours with either 500ng/ml anti-Fas agonistic CH11 mAb or 100ng/ml TRAIL before analysis.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total cellular RNAs were isolated using Trizol reagent (Invitrogen). One µg of total RNA was subjected to RT in a 20 µl reaction volume using oligo(dT) primer and M-MLV reverse transcriptase (Invitrogen) according to the manufacturer's protocol. Three µl of the RT reaction was then amplified by PCR for 30 cycles using the following conditions: 94°C for 30 sec ; 58°C for 1 min and 72°C for 2 min. The primers used were: flip short forward

(LegendsFigure 1 :

 1 Figure 1: E2F1 induces apoptosis in lung adenocarcinoma cells through a caspase-8-and

Figure 2 :

 2 Figure 2: E2F1 activates caspase-8 at the DISC level

Figure 3 :

 3 Figure 3: The short isoform of c-FLIP is a downstream target of E2F1

Figure 4 :

 4 Figure 4: E2F1 specifically downregulates c-flip s mRNA

Figure 5 :

 5 Figure 5: E2F1 sensitizes NSCLC cells to death receptor-mediated apoptosis by

Figure 6 :

 6 Figure 6: E2F1 enhances tumor cells sensitivity to lymphocyte T-dependent cytotoxicityT cell-dependent cytotoxicity was measured in the presence of EGTA using a 24 hours51 Cr-

Figure 7 :

 7 Figure 7: Western blot analysis of E2F1 and c-FLIP expression in human lung

Acknowledgements

We thank Dr. K. Helin and Dr. J. Nevins for providing plasmids and cells. We also thank Dr. Alicia Lajmanovitch (INSERM, EMI353) for hepful discussions. This work was supported by a grant to E.B. from La Ligue Nationale Contre le Cancer as an «équipe labellisée» and by the Region Rhône Alpes (Thématique prioritaire cancer and Canceropole 2003: Oncocell, Epimed and INACancer). C.S. was supported by a "poste accueil" INSERM. We thank Celine Lampreia and Pascal Perron for technical assistance.