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Oncogene (2008) 27, 1545–1553

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ORIGINAL ARTICLE

The human carcinoembryonic antigen (CEA) GPI anchor mediates anoikis inhibition by inactivation of the intrinsic death pathway P Camacho-Leal1 and CP Stanners Department of Biochemistry and McGill Cancer Center, McGill University, Promenade Sir-William Osler, Montreal, Quebec, Canada

Human carcinoembryonic antigen (CEA) is a cell surface adhesion molecule member of the Immunoglobulin Superfamily (IgSF). Aberrant upregulation of CEA is a common feature found in a wide variety of human cancers such as colon, breast and lung. Previous in vitro and in vivo results have demonstrated that CEA can have tumorigenic effects including the inhibition of cell differentiation and anoikis, a specific type of apoptosis triggered by the absence of extracellular matrix–cell contacts. In the present work, we investigate the involvement of the caspase cascade in CEAmediated inhibition of anoikis and the structural requirements for this signal. Expression of CEA and/or a chimeric protein consisting of the NCAM extracellular domain attached to the CEA-GPI anchor correlates with an early inactivation of caspase-9 and activation of the PI3-K/Akt survival pathway, and at later times, inactivation of caspase-8. The CEA-mediated caspase inactivation as well as activation of Akt was not observed by expression of a CEA molecule incapable of self-binding (DNCEA). These results suggest that the intrinsic caspase pathway is involved in the inhibitory effects of anoikis by CEA and this signal is dependent on the presence of self-adhesive extracellular domains and a CEA-GPI anchor. Oncogene (2008) 27, 1545–1553; doi:10.1038/sj.onc.1210789; published online 24 September 2007 Keywords: CEA; anoikis; molecular mechanism; caspases

Introduction Cell attachment to specific extracellular matrix (ECM) components has been shown to induce signal transduction cascades inside the cell that ultimately control cell growth, differentiation, and cell death (Giancotti and Ruoslahti, 1999). Disruption of cell–ECM contacts triggers a specific type of apoptosis termed anoikis Correspondence: Professor CP Stanners, Department of Biochemistry and Cancer Center, McGill University, 3655 Promenade Sir-William Osler, Montreál, Que´bec, H3G 1Y6 Canada. E-mail: [email protected] 1 Current address: Dipartimento di Genetica, Biologia e Biochimica, Molecular Biotechnology Center, Universita di Torino, Via Nizza 52, 10126, Torino, Italy. Received 26 April 2007; revised 31 July 2007; accepted 1 August 2007; published online 24 September 2007

(Frisch and Screaton, 2001; Reddig and Juliano, 2005) which has been observed in various types of adherent cells such as intestinal (Grossmann, 2002), epidermal (Tiberio et al., 2002; Marconi et al., 2004), fibroblast (Tian et al., 2002; Nho et al., 2005) and prostate cells (Rennebeck et al., 2005). The anchorage of cells to ECM is mainly mediated by integrin receptors leading to a ligand-dependent activation of two major downstream signaling pathways implicated in cell survival, the Raf/MAPK and the PI3-K/Akt cascades (Frisch and Ruoslahti, 1997; Stupack and Cheresh, 2002). As in most apoptotic pathways, anoikis protection by integrins has been shown to be controlled by regulation of the caspase cascade (Gilmore, 2005), although some studies have demonstrated the absence of caspase activation during early events in the anoikis program (Wang et al., 2003). Integrin-dependent caspase regulation occurs via both the intrinsic and extrinsic death pathways (Frisch, 1999; Rytomaa et al., 2000; Grossmann, 2002; Marconi et al., 2004). Whereas the intrinsic pathway involves mitochondrial permeabilization and release of cytochrome c, leading to caspase-9 activation (Boatright and Salvesen, 2003), the extrinsic death pathway results in activation of caspase-8 and -10 (Boatright and Salvesen, 2003). GPI-anchored carcinoembryonic antigen (CEA) family members, CEA and CEACAM6, belong to the human CEA family and are normally mainly expressed in the gastrointestinal tract (Stanners and Fuks, 1998; Hammarstrom, 1999), but are overexpressed in as many as 70% of all human cancers (Rosenberg et al., 1993; Nollau et al., 1997). It has been demonstrated that essentially all CEA family members, including GPIanchored CEA and CEACAM6, function as homotypic intercellular adhesion molecules in vitro (Benchimol et al., 1989; Oikawa et al., 1989; Stanners and Fuks, 1998). In contrast with the transmembrane member CEACAM1 (Eidelman et al., 1993; Rojas et al., 1996; Ilantzis et al., 2002; Taheri et al., 2003), CEA and CEACAM6 overexpression inhibits cell differentiation and anoikis in vitro (Eidelman et al., 1993; Ordonez et al., 2000; Screaton et al., 2000; Soeth et al., 2001; Taheri et al., 2003; Duxbury et al., 2004) and in vivo (Chan et al., unpublished results). The structural requirements for the inhibitory effects of CEA on differentiation were demonstrated to depend critically on the CEA-specific GPI anchor attached to self-binding

Molecular mechanism of CEA-mediated anoikis inhibition M del Pilar Camacho-Leal and CP Stanners

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extracellular domains (Screaton et al., 2000; Taheri et al., 2003). Subsequent findings showed that activation of the integrin a5b1 was involved in these inhibitory effects (Ordonez et al., 2007) and that this activation results from co-clustering of integrin molecules upon CEA clustering in the same membrane microdomains (Camacho-Leal et al., 2007). Associated integrin-specific signaling elements, such as those involved in the PI3-K and the MAPK signaling pathways, were also activated upon CEA cross-linking (Camacho-Leal et al., 2007). In the present work, we initiated a study to define the molecular mechanism by which CEA mediates its inhibitory effects on anoikis and the structural requirements for these effects. We found that, as with differentiation inhibition, self-adhesive extracellular domains linked to a CEA-specific GPI anchor are required for anoikis inhibition. The intrinsic caspase signaling pathway involving PI3-K and Akt activation was implicated in the blockage of anoikis by CEA. We observed that CEA prevented initially the activation of caspase-9 and the release to the cytoplasm of cytochrome c and, at later times, inhibited the activation of caspase-8 and -3. Pharmacological inhibition of PI3-K but not MAPK could partially reverse the CEAmediated inhibition of caspase activation. These results suggest that CEA induces an early inactivation of the intrinsic apoptotic pathway mediated by the inhibition of the PI3-K/Akt survival pathway. Results Structural requirements for inhibition of anoikis by CEA To determine whether, as previously demonstrated for differentiation inhibition (Screaton et al., 2000; Taheri et al., 2003), the presence of the CEA GPI anchor attached to a self-binding extracellular domain was required to confer anoikis resistance, we used the N-C chimeric protein, consisting of a CEA GPI-anchor fused with the self-binding external domain of Neural Cell Adhesion Molecule (NCAM), another immunoglobulin superfamily member which normally allows cell differentiation to occur (Screaton et al., 2000), and a CEA mutant DNCEA, lacking a major portion of the N-domain of CEA (DNCEA) (Taheri et al., 2003). L6 myoblasts were cultured in suspension for 36 h, as previously described (Ordonez et al., 2000), and the proportion of apoptotic cells was determined (Figure 1a). As shown previously, CEA transfectant cells were less prone to undergo anoikis than parental cells (Ordonez et al., 2000). A reduced proportion of apoptotic cells was found in cell populations expressing Bcl-2, which has been shown to have antiapoptotic effects under conditions that induce anoikis (Frisch and Screaton, 2001); this transfectant was used as a positive control for all experiments. TUNEL staining of cells expressing DNCEA, CEACAM1, NCAM, or the parental cells, showed higher levels of anoikis induction, whereas cells expressing the chimeric protein N-C inhibited apoptosis, as observed in cells expressing CEA itself (Figure 1b). Oncogene

Activation of cysteine protease caspase-3 is a hallmark of apoptosis, and its activation has been implicated as part of the anoikis cascade (Reddig and Juliano, 2005). We therefore evaluated caspase-3 activation by detection of its cleaved product (17 kDa) in attached L6 transfectant cells and in cells incubated in suspension for 12, 24 (Figure 1c) and 48 h (data not shown). Western blot analysis detected the full-length 35 kDa caspase-3 molecule in attached cells, whereas cleaved 17 kDa caspase-3 product was detected in parental L6 cells as early as 12 h after anoikis induction. Cleavage was evident in many of the transfectants after 24 h. Almost complete proteolysis of caspase-3 was observed at 48 h for all the cell lines (data not shown). Densitometric analysis of the western blots was used to determine the percentage of cleaved caspase-3 fragment with respect to the total caspase levels (full-length caspase, plus cleaved fragment, Figure 1c). The latter results were consistent with the TUNEL assay data, since CEA and N-C expressing cells showed less caspase-3 cleavage and presumably caspase-3 activation, in contrast to NCAM, CC1 and DNCEA transfectants. CEA expression prevents the release of cytochrome c and the activation of caspase-9 Caspase-3 activity represents a late event in the apoptotic process, since activation of initiator caspase2, -8, -9 and -10 is required for caspase-3 activation (Boatright and Salvesen, 2003). In order to determine whether caspase-9 activation was affected by CEA, we evaluated the activation of caspase-9 after 12 and 24 h of cell incubation in suspension both by western blot analysis (Figure 2a) and activity by a spectrophotometric assay (Figures 2b and c). Western blot analysis showed that CEA expression and the presence of the CEA-GPI anchor in N-C but not NCAM, CC1 or DNCEA expression, markedly inhibited caspase-9 activation at 24 h after anoikis induction (Figure 2a), since the appearance of cleaved caspase-9 fragment (17 kDa) was partially prevented in CEA and N-C expressing L6 cells. In contrast to caspase-3, caspase-9 activation was evident at 12 h after anoikis induction (Figure 2a). In agreement with the latter results, a reduced caspase-9 activity was also observed at the same time points upon CEA and N-C expression (Figures 2b and c). The release of cytochrome c from the mitochondria into the cytosol is a hallmark of apoptosis induced by caspase-9 activation. Since we observed that expression of CEA inhibited caspase-9 activity, we decided to explore the involvement of cytochrome c release as a possible part of the apoptotic mechanism inhibited by CEA. Cytosolic fractions were obtained and cytochrome c levels evaluated by western blot analysis in all transfected cell lines. Figure 2d shows, as expected, no cytochrome c release into the cytosol in attached cells whereas, after 12 h of anoikis induction, cytochrome c was detected, but at markedly lower levels, for Bcl-2, CEA or N-C expressing cells. Released cytochrome c levels were shown to increase after 24 h of anoikis induction (Figure 2d). These results demonstrate that


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Figure 1 CEA-GPI anchor and self-adhesive extracellular domains are required for CEA-mediated anoikis inhibition via inactivation of caspase-3. Apoptotic L6 parental and transfected cells detected by positive staining using the TUNEL assay on cell populations suspended for 36 h in ultra-low attachment hydrogel plates (a). The apoptotic index was calculated by scoring the percentage of Tunelpositive cells with respect to the total cell count (7s.d.; *Po0.006) (b). Western blot analysis of caspase-3 cleavage product (c) from suspended L6 transfectants after 12 and 24 h under detachment conditions. Histogram shown in (c) represents the densitometry analysis of caspase-3 western blot at 24 h under detachment conditions. These results are representative of three independent experiments.

CEA expression inhibits the intrinsic death pathway by preventing cytochrome c release and the subsequent activation of caspase-9. These effects correlated with the presence of the CEA GPI-anchor and self-adhesive extracellular domains. Caspase-8 inactivation induced by CEA occurs later in the apoptotic process During the extrinsic death pathway, ligation of death receptors such as TNF or Fas on the cell surface promotes the assembly of the death-inducing signaling complex (DISC). This complex recruits and activates caspase-8 via the death receptor FADD. Some studies have shown that FADD and an early activation of caspase-8 are involved in the anoikis process (Frisch, 1999). In some cases, crosstalk

between the intrinsic and extrinsic pathways occurs upon caspase-8 activation, through the Bcl-2-like protein Bid and subsequent activation of cytochrome c (Stupack and Cheresh, 2002). We therefore decided to explore the contribution of the extrinsic death pathway during the CEA-mediated block of anoikis. Activity assays for caspase-8 were performed after 12 and 24 h of anoikis induction. In contrast to the effects of CEA and N-C expression observed on caspase-9 at 12 h of suspension culture (Figure 2b), smaller changes in caspase-8 activation were observed at the same time point, whereas only after 24 h a higher magnitude differences were seen for caspase-8 activity (Figure 3b). Caspase-8 activation was partially prevented by CEA and N-C expression whereas control NCAM, CC1 and DNCEA expression showed no such inhibition of caspase-8 activity levels (Figure 3b). The latter Oncogene


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Figure 2 CEA inhibits the intrinsic death pathway through inhibition of caspase-9 activation and cytochrome c release. Caspase-9 cleavage product was detected by immunoblot analysis of lysates from attached cells, and from suspended cells 12 and 24 h after detachment (a). Histogram shown in (a) represents the densitometry analysis of caspase-9 western blot at 24 h under detachment conditions. Caspase-9 activity was measured in suspended L6 transfectants after 12 h (7s.e.; *Po0.003) (b) and 24 h (7s.e.; *Po0.0001) (c) of anoikis induction. In (d), cytochrome c levels were evaluated in cytosolic fractions from attached L6 transfectants and from such cells after 12 and 24 h in suspension. Histogram shown in (d) represents the densitometry analysis of cytochrome c western blot at 12 h under detachment conditions. Means of three independent experiments are shown (b, c) and representative results out of three independent experiments are shown in (a, d).

results thus demonstrate that CEA also inhibits the activation of caspase-8 induced by loss of anchorage and that this inhibition seems to occur late in the apoptotic process, presumably after inhibition of caspase-9 activation and cytochrome c release. Inhibition of the PI3-K cascade but not MAPK reverses the caspase-9 inactivation induced by CEA expression We previously demonstrated that activation of the PI3K/Akt and MAPK signaling cascades are involved in the integrin activation induced by crosslinking of NCEA Oncogene

(Camacho-Leal et al., 2007). We therefore investigated the status of Akt and MAPK after inducing anoikis by suspension culture. Transfectant L6 cell lysates obtained from attached cells and cells cultured in suspension were evaluated for Akt phosphorylation at ser 473 with a specific anti-phospho ser 473 Akt polyclonal Ab by western blot analysis (Figure 4). As observed previously, Akt phosphorylation levels remained unchanged in attached cells regardless of CEA expression (Figure 4). After 12 h under detachment conditions, the levels of phosphorylated Akt were higher for Bcl-2, CEA and N-C expressing cells than for parental L6 cells and for


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Figure 3 CEA-mediated inhibition of caspase-8 activation occurs late in the anoikis program. Activity assays for caspase-8 were evaluated in 12 h (7s.e.; *Po0.0009) (a) and 24 h (7s.e.; *Po0.003) (b) suspended L6 transfectant cultures. The results are the mean of three independent experiments.

DNCEA, NCAM and CC1 transfectants. No effects were seen for MAPK phosphorylation (data not shown). These results suggest that Akt activation may be implicated in the anoikis inhibition mediated by CEA. To test for a possible role of the PI3-K/Akt signaling pathway in the CEA-mediated caspase-9 and -3 attenuation associated with anoikis inhibition, the effect of the specific inhibitor of PI3-K, LY29002, was evaluated. Cells were treated for 24 h under suspension conditions with 50 mM of LY29002 or, as a specificity control, 30 mM of the MAPK-specific inhibitor PD098059; cell lysates were then evaluated for caspase-9 (Figure 5a) and caspase-3 cleavage (Figure 5b) by western blot analysis using specific caspase mAbs. Densitometric analysis of caspase-9/-3 western blots, giving the percentage of caspase-3/-9 cleaved fragment with respect to the total caspase levels, demonstrated that cell treatment with LY29002 could partially reverse the caspase-9/-3 inactivation induced by CEA and N-C expression. No effects were observed for the PD098059 inhibitor. It is interesting to note that the Bcl-2-mediated caspase block was not prevented by inhibition of either of these signaling pathways. Thus, the latter results suggest that activation of the PI3-K/Akt pathway is involved in the CEA-mediated inactivation of caspase-9 and -3, although the fact that the PI3-K/Akt inhibition only partially reversed the effect of CEA, suggests that additional survival signals may be involved in CEA-mediated anoikis inhibition.

Discussion The inhibitory role of CEA in cell differentiation (Eidelman et al., 1993; Screaton et al., 1997; Taheri et al., 2003) has been extensively documented. Structural studies indicated that the CEA-specific GPI-anchor attached to a self-binding external domain was necessary

Figure 4 Activation of Akt correlates with CEA expression during anoikis. Total Akt and Akt phosphorylated on Ser 473 were evaluated by western blot analysis in attached L6 transfectant cells and after 12 h of anoikis induction in suspended cells. This experiment is representative of three independent experiments.

and sufficient for its inhibitory effects on cell differentiation (Screaton et al., 2000; Taheri et al., 2003). In the case of anoikis inhibition, although it is well documented that CEA/CEACAM6 expression can inhibit the anoikis of human colorectal carcinoma cells (Ordonez et al., 2000; Soeth et al., 2001) and human pancreatic carcinoma cells (Duxbury et al., 2004), an effect that it is not produced by the transmembraneanchored CEA-family member CEACAM1 or the GPIanchored variant of the immunoglobulin superfamily member adhesion molecule, NCAM, (Ordonez et al., Oncogene


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Figure 5 PI3-K inhibition partially reverses the CEA-mediated inhibition of caspase-9 and -3 activity. Caspase-9 (a) and caspase-3 (b) cleavage products were detected by western blot analysis after treatment with the PI3-K inhibitor, LY294002, at 50 mm or the MAPK inhibitor, PD098059, at 30 mm for 24 h under detachment conditions. Histograms represent densitometric analysis of caspase-3 and -9 western blots. Results shown are representative of three independent experiments.

2000), the molecular mechanism implicated in this inhibitory effect was unclear. This study was focused on the molecular mediators associated with the anoikis resistance conferred by CEA expression. Although in some cases early activation of the anoikis program can occur in the absence of caspase activation (Wang et al., 2003), the latter mechanism represents an important event involved in anoikis regulation (Frisch and Screaton, 2001; Stupack and Cheresh, 2002; Gilmore, 2005). Recently, the increase in anoikis susceptibility of CEACAM6-expressing human pancreatic carcinoma cells produced by CEACAM6 siRNA gene silencing was shown to be associated with an increase in caspase-8 and -3 activities (Duxbury et al., 2004). Our findings indicate that, in the case of CEA expression, there is also an associated caspase-mediated anoikis resistance, since we observed a partial inactivation of caspase-3 (Figure 1c) which correlated with anoikis inhibition by CEA (Figure 1a). Although CEA Oncogene

expression partially induced the inactivation of caspase8 (Figure 3), our data support a more prominent contribution of the intrinsic pathway, since we observed a CEA-mediated inhibition of cytochrome c release from the mitochondria into the cytosol (Figure 2d) and an inactivation of caspase-9 (Figures 2a–c), whereas caspase-8 inactivation occurred later during the anoikis process (Figure 3). It is known that crosstalk between the intrinsic and extrinsic pathways occurs through cleavage of the BH3-only protein Bid, generating the truncated form of Bid, t-Bid (Li et al., 1998; Luo et al., 1998; Deng et al., 2002). t-Bid then translocates to the outer mitochondrial membrane and promotes oligomerization of Bax or Bak to facilitate release of cytochrome c and subsequent caspase-9 activation (Li et al., 1998; Luo et al., 1998). In addition, a caspase-9-mediated caspase-8 activation has been reported in certain cell systems (Cha et al., 2001; Viswanath et al., 2001). Although our results show clearly the involvement of


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both caspase pathways in the anoikis inhibition induced by CEA, further studies are required to establish whether caspase-8 inactivation represents an independent pathway to that of caspase-9, or crosstalk between these two pathways gives a caspase-9-dependent inhibition of caspase-8 activity. Previous structural studies of the CEA molecule demonstrated that the CEA-GPI anchor and homophilic binding of its extracellular domains were required to inhibit cell differentiation (Screaton et al., 2000; Taheri et al., 2003). In this work, results obtained with the GPI-anchored isoform of NCAM, the N-C chimeric protein (the NCAM external domain attached to the CEA GPI anchor) and the DNCEA mutant (with a deletion in the external domain preventing self-binding) showed that these structural requirements were also applicable for the CEA-mediated anoikis inhibition (Figure 1). Thus the inactivation of caspase-3 (Figure 1), -9 (Figure 2) and -8 (Figure 3) induced by CEA expression was also induced by the expression of N-C, whereas the expression of the DNCEA mutant had no such effect. Our previous evidence supported a model for CEA signaling in which clustering of CEA GPI anchors in lipid rafts leads to a5 integrin clustering and activation followed by activation of the PI3-K/Akt and MAPK signaling pathways (Camacho-Leal et al., 2007). Indeed, in colonic cells, we have shown that perturbation of a5 integrin–ECM interaction reversed the CEA-mediated anoikis inhibition (Ordonez et al., 2007). The role of the PI3-K/Akt and MAPK cascades in cell survival mechanisms has been extensively documented (Frisch and Screaton, 2001). In addition, integrin activation has been shown to represent an important event in the regulation of the intrinsic and extrinsic death signals (Stupack and Cheresh, 2002). In this study, we found that expression of CEA induced Akt activation during anoikis, and that this effect had the same structural requirements as CEA-mediated anoikis inhibition, that is, required the presence of the CEA GPI-anchor and was not induced by the expression of the DNCEA mutant (Figure 4). Furthermore, pharmacological inhibition of PI3-K but not MAPK, could partially reverse the inactivation of caspase-9 and -3 induced by CEA (Figure 5), although this effect was not completely reversed, suggesting the involvement of other survival signals in the CEA-mediated anoikis inhibition. In summary, the current study provides significant insight into the molecular mechanism involved in the inhibitory effects of CEA on anoikis. We provide evidence demonstrating that the latter inhibition occurs in a caspase-dependent fashion by early inactivation of the intrinsic pathway, and a subsequent block of caspase-8 activity. The activation of the PI3-K pathway was also shown to be implicated in the CEA-mediated inhibitory effects. We propose that inactivation of the intrinsic death pathway is involved in the CEA-mediated anoikis inhibition and that, as with differentiation inhibition, this effect requires the CEA-GPI anchor attached to self-binding extracellular domains.

Materials and methods Cell lines and culture conditions L6 rat myoblasts (Yaffe, 1968) were grown in monolayer cultures in Dulbecco’s modified Eagle’s media (DMEM) containing 10% fetal bovine serum (FBS, GIBCO BRL, Burlington, ON, Canada) supplemented with 100 mg ml 1 streptomycin and 100 U ml 1 penicillin (GIBCO BRL) (growth medium: GM). Cultures of subconfluent proliferating L6 rat myoblast cells were used for all experiments. To induce anoikis, 1.5 ml of suspended cells at 2 105 cells ml 1 were cultured in GM using ultra-low cell attachment hydrogel plates (Corning Lowell, MA, USA) for the indicated times. Cell transfection and infection Transfection procedures were carried out as previously described (Eidelman et al., 1993; Screaton et al., 2000). For details see Supplementary Information. Caspase activity assays A caspase-8 and -9 colorimetric activity kit from CHEMICON (Temecula, CA, USA). was used to determine the activity of each one of these caspases. Briefly, after 12 or 24 h of culturing L6 cells in suspension, 2 106 L6 transfectant cells were resuspended in 250 ml of chilled 1 cell lysis buffer provided with the kit. Cells were incubated on ice for 10 min and then centrifuged for 5 min. Supernatant was collected (cytosolic extract) and protein concentration was determined by the Bradford protein assay (Pierce, Nepean, ON, Canada). Between 50 and 100 mg of protein was incubated for 2–3 h at 37 1C with either caspase-8 or -9 substrate (assays for caspases-8 and -9 were performed simultaneously for each experiment). Samples were read at 405 nm in a microtiter plate reader. Background readings of the substrate sample alone were subtracted from the readings of experimental samples. Preparation of cytosolic fractions for cytochrome c Cytosolic fractions were obtained as previously described (Rytomaa et al., 2000). For details see Supplementary Information. Terminal deoxynucleotidyl transferase (TdT) nick end labeling (TUNEL) Apoptotic cells from suspension cell cultures of L6 transfectants were detected by the TUNEL assay according to the protocol provided by the APOAlertt DNA Fragmentation Assay Kit from BD Biosciences, Bedford, MA, USA. Apoptotic cells were visualized using a Nikon TE300 microscope with a Bio-Rad Radiance 2000 confocal accessory. As a positive control for detection of DNA fragmentation, cells were treated with DNAse I before TUNEL. As a negative control, terminal transferase was omitted from the TUNEL reaction. The apoptotic index was calculated by scoring the percentage of apoptoticTUNEL positive cells with respect to the total cell count (200–300 cells). Western blot analysis Western blot analyses of total cell lysates or cytosolic fractions were carried out as previously described (CamachoLeal et al., 2007). Oncogene


1552 Statistical analysis Statistical comparison of means between groups was performed using the two-tailed unpaired Student’s t-test. Po0.05 was considered significant. Antibodies and inhibitors used are included in Supplementary Information.

Acknowledgements This work was supported by a grant from the Canadian Institutes of Health Research. MPC-L was supported by the National Science Council (CONACYT), Mexico.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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