Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells

Experimental Cell Research 311 (2005) 106 – 116 www.elsevier.com/locate/yexcr

Research Article

Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells Benjamin D. Ne´el a,1, Abdel Aouacheria1, 2, Anne-Laure Nouvion a, Xavier Ronot b, Germain Gillet a,* a IBCP, UMR 5086 CNRS/Universite´ Claude Bernard, IFR 128, 7 passage du Vercors, F69367, Lyon cedex 07, France Dynamique Cellulaire, Ecole Pratique des Hautes Etudes, IFRT 130, UMR CNRS 5525 Universite´ Joseph Fourier, 38706 La Tronche cedex, France

b

Received 26 April 2005, revised version received 20 August 2005, accepted 1 September 2005 Available online 3 October 2005

Abstract Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60v-src tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60v-src , we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60v-src inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin – proteasome pathway is recruited early after p60v-src inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells. D 2005 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Calpains; Caspases; Cytoskeleton; Proteasome; Retina; v-src

Introduction Apoptosis is critical for tissue homeostasis. Apoptotic cells exhibit typical morphological and biochemical changes, including chromatin condensation, nuclear fragmentation, DNA laddering and cell shrinkage. Moreover, in the case of adherent cells, major morphological changes occur, which are eventually followed by detachment from the substratum. Such apoptotic processes are accompanied by the activation of caspases and other proteases, including calpains and the proteasome [1 –3].

* Corresponding author. Fax: +33 4 72 72 26 01. E-mail address: [email protected] (G. Gillet). 1 These authors contributed equally to this work. 2 Present address: Biome´trie et Biologie Evolutive, UMR 5558 CNRS/ Universite´ Claude Bernard, 43 Boulevard du 11 novembre, F69622 Villeurbanne cedex, France. 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.09.001

Calpains are Ca2+-activated proteases involved in the degradation of proteins during apoptosis [4]. They are localized to focal adhesions [5], where a growing number of calpain substrates have been identified, including structural proteins, such as talin and the integrin h3 subunit [6,7], as well as proteins involved in signal transduction networks, such as protein kinase C, focal adhesion kinase (FAK), p60v-src and protein tyrosine phosphatase-1B [8]. The role of caspases in apoptosis has been clearly established [9], whereas much less is known about the exact role of the ubiquitin – proteasome pathway. In particular, proteasome inhibitors have been shown to induce or inhibit apoptosis, depending on cell type [10]. To date, a comprehensive view of the protease intracellular network is still lacking. As these proteases are potential drug targets, it is important to evaluate the extent to which these enzymes are deregulated in cancer cells.

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V-src-transformed cells have been widely used as a model for the study of neoplastic transformation. Expression of the v-src oncogene is associated with deregulated growth and loss of cell adhesion [11]. Rous sarcoma virus (RSV)-transformed quail neuroretina cells (QNR cells) are used as a model to study neoplastic transformation in nerve cells [12,13]. These cells, although fully transformed, still exhibit typical features of differentiated nerve cells [14 – 16]. We previously made use of QNR cells (QNR-ts68) transformed by a thermosensitive (NYts68) strain of RSV to compare the combined effects of serum and the p60v-src tyrosine kinase in nerve cells [13]. We showed that inactivation of p60v-src resulted in dramatic cell shape changes followed by detachment from the substratum and apoptosis, these effects being enhanced in the absence of serum [17]. Together, these results highlight the tight control exerted by p60v-src on cell morphology and apoptosis. Here, we used a pharmacological approach to evaluate the respective roles of the major protease cascades in the response of QNR cells to p60v-src inactivation.

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Results and discussion

addition to these morphological changes, a number of apoptotic cells began to detach from the substratum after 9– 11 h (Fig. 1B, 41.5-C S, d1). Apoptosis was observed much later at a higher cell density of 106 cells per dish (Fig. 1B, 41.5-C S, d2), while at an even higher density of 2.106 cells per dish (Fig. 1B, 41.5-C S, d3), cells exhibited typical migration and neurite outgrowth within 12 – 18 h, without significant apoptosis. Cytoplasmic projections fused together and formed ‘‘connecting bridges’’ between cellular aggregates (Fig. 1B, 41.5-C S, d3). This suggested that QNR-ts68 cells that could not make contact with neighboring cells would die, while cellular aggregates remained viable even under unfavorable conditions. Thus, cell –cell contacts, possibly mediated by adhesion molecules such as cadherins, would activate anti-apoptotic signaling pathways, preventing the activation of caspase-3 and subsequent cell death that follow p60v-src inactivation [17]. Indeed, sensitivity to apoptosis, as measured by detecting phosphatidyl serine exposure at the outer leaflet of the plasma membrane, was found to be inversely correlated with cell density (Fig. 2). In summary, following p60v-src denaturation, cell shape remodeling begins after a latency period; such morphological changes are followed by apoptosis, which depends both on cell density and serum concentration.

Analysis of QNR-ts68 behavior following p60v-src inactivation

Involvement of the ubiquitin – proteasome in v-src-induced cell remodeling

At permissive temperature (36.5-C), quail neuroretina cells infected either by the thermosensitive RSV strain tsNY68 (QNR-ts68 cells) or by the wild type strain SRA (QNR-SRA cells) are highly refringent and exhibit a typical transformed phenotype (Fig. 1A, see also [12,13,18]). When transferred at non-permissive temperature (41.5-C), QNRts68 cells show profound morphological remodeling, increased cell – cell contacts and apoptosis (Fig. 1A). These changes are closely correlated with the thermal inactivation of p60v-src [17]. In contrast, the kinase activity of p60v-src is temperature-independent in QNR-SRA cells. These latter cells are thus still fully transformed, showing no morphological change when grown at 41.5-C (Fig. 1A, see also [17]). In the same way, we previously showed that the temperature shift does not affect the cell cycle or the apoptosis rate in QNR-SRA, contrary to QNR-ts68 cells [13,17]. Therefore, in addition to the cell cycle and apoptosis, the morphology of RSV-infected QNR cells is profoundly affected by the activity of the p60v-src tyrosine kinase. The behavior of QNR-ts68 cells following p60v-src denaturation at restrictive temperature (41.5-C) was analyzed by time-lapse videomicroscopy as a function of cell density and serum concentration. In the presence of 10% serum, when seeded at a density of 0.5.106 cells per 35-mm dish, QNR cells rapidly flattened, becoming less refringent within 6 –8 h, (Fig. 1B, 41.5-C + S, d1). When serum was withdrawn at the same time as the shift up to 41.5-C, in

Proteolytic activities participate in cell shape control in a number of cells [3,19,20]. QNR-ts68 cells undergo major morphological changes following p60v-src thermal inactivation (Fig. 1). In presence of high serum concentration (10%), these changes occur 6 –8 h following the temperature shift, apoptotic cells becoming visible 14 –16 h later (Fig. 1B, see also [13,17]). The number of apoptotic cells increases significantly when the temperature shift up to 41.5-C is carried out in absence of serum [17]; Fig. 3 shows that, in these latter conditions, p60v-src thermal inactivation results in the marked activation of caspase-3. In contrast, at 36.5-C, i.e. when p60v-src is active, serum removal does not induce cell death, while no activation of caspase-3 can be detected (Fig. 3, see also [17]). In addition to caspase-3, we detected calpain and proteasome-like activities in QNR-TS68 cells using in vitro assays (Fig. 4). A series of inhibitors were used to assess the nature of these protease activities. As shown in Fig. 4, both activities were inhibited by ALLN, an inhibitor of proteasome and calpains [21,22], while lactacystin (CLBL) specifically inhibited the proteasome-like activity. Moreover, the three calpain inhibitors E64, calpeptin and calpain inhibitor IV specifically inhibited the calpain-like activity (Fig. 4B). Finally, the serine protease inhibitor TPCK and the caspase inhibitor ZVAD had not effect on the detected protease activities. Together, these results show that caspases, calpains and proteasome are actually present in QNR-ts68 cells. Moreover, some of these proteases appear

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to be regulated by p60v-src . Indeed, p60v-src inactivation resulted in both the activation of caspase-3 (Fig. 3) and the partial inactivation of the proteasome (Fig. 4A). In contrast, calpains do not depend on the status of p60v-src in QNR-ts68 cells (Fig. 4B). To determine which proteases might be involved in cell shape remodeling induced by p60v-src inactivation, cells grown in presence of 10% serum were shifted from 36.5-C up to 41.5-C with or without various enzyme inhibitors. In such conditions, incubation with Z.VAD.FMK (Fig. 5A) or the serine protease inhibitors TPCK and TLCK (not shown) had little effect on cell morphology, i.e. did not prevent flattening and loss of refringency observed at 41.5-C. However, cell density at 41.5-C was significantly higher in the presence of Z.VAD.FMK, due to its antiapoptotic effect (not shown). Treatment of cells with ALLN prevented the morphological changes usually observed after p60v-src denaturation. Indeed, under these latter conditions, cells were still highly refringent, exhibiting typical transformed morphology. In contrast, none of the calpain inhibitors (E64, calpain inhibitor IV and calpeptin)

did impair cellular remodeling (Fig. 5A), suggesting that the effect of ALLN was due to the inhibition of the proteasome, but not of calpains. Alternatively, the lack of effect of the calpain inhibitors could be due to their inability to cross the plasma membrane and inactivate calpains in vivo. To address this point, we checked the effect of these inhibitors using a calpain assay directly performed into live cells. As shown in Fig. 5B, calpain inhibitor IV, calpeptin, ALLN, and to a lesser extent E64, but not CLBL, were actually found to inhibit calpains in QNR-ts68 cells in vivo. These observations indicate notably that, despite its poor ability to diffuse across biological membranes, the uptake of E64 by QNR cells is sufficient to inhibit intracellular calpains, although less efficiently compared to ALLN, calpain inhibitor IV or calpeptin. Actually, E64 exhibits in vivo effects in a number of cell types [23 – 25], including nerve cells [26], although generally at much higher concentration compared to cell-permeable inhibitors. In this respect, Wilcox and Mason suggested that E64 might enter into the cells by pinocytosis [27]. Thus, taken together, the results depicted

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Fig. 1. Effect of p60v-src thermal inactivation in ts68-QNR cells. (A) All cells were seeded at a density of 106 cells per 35 mm per dish. Upper six panels: phase contrast microscopy showing ts68-infected QNR cells (left) and SRA-infected QNR cells (right) cultured at permissive temperature in presence of 10% serum (36.5-C + S) and after 36 h at a non-permissive temperature in presence (41.5-C + S) or absence (41.5-C S) of serum. At 41.5-C, QNR-ts68 cells are flattened and much less refringent, with increased cell – cell contacts. Numerous round apoptotic cells are observed in absence of serum (41.5-C S). In contrast, the morphology of QNR-SRA cells is unchanged at 41.5-C; in addition, SRA-infected cells are resistant to serum withdrawal. Bottom panel (differential phase microscopy): under the latter conditions, only interconnected QNR-ts68 cells survived after a prolonged incubation time at restrictive temperature (41.5-C S, 72 h). (B) Time-lapse videomicroscopy. Cells were seeded at various densities (35 mm dishes) and grown overnight at 36.5-C with 10% serum. Time-lapse video microscopy recordings were then made of cells shifted up to 41.5-C with or without serum. Images show representative fields scanned at regular intervals after the temperature shift. The time (hours) is shown on each panel. (41 + S, d1): After a 6-h-long latency phase, cells cultured at 41.5-C with 10% serum at a low density (0.5.106 cells per dish) became flattened and less refringent, with numerous intercellular contacts. A small number of apoptotic cells are observed. (41 S, d1): Cells cultured at 41.5-C in the absence of serum at the same density (0.5.106 per dish) rounded rapidly, exhibiting morphological features of cell death 7 h – 9 h after the temperature shift, massive cell death occurring at 14 – 15 h. (41 S, d2): When seeded at an intermediate density (106 cells per dish), apoptotic cells being detected much later (14 – 16 h). (41 S, d3): At high density (2.106 cells per dish), cells extended cytoplasmic projections or moved and formed aggregates, and few apoptotic cells were observed.

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proteasome by CLBL does not induce by itself these cell shape changes but, in contrast, totally prevents them (see Fig. 5A); this clearly indicates that morphological changes require at least a baseline level of proteasome activity. In this respect, a precise time course analysis would be helpful to decipher whether the down-regulation of proteasome activity occurs upstream or downstream of cell shape changes following p60v-src inactivation. In summary, our results show that, following p60v-src inactivation, the proteasome, but not calpains or caspases, plays a central role in the morphological changes shown by the QNR-ts68 cells, which are associated with the loss of the transformed phenotype [17]. Involvement of caspases, but not calpains or the proteasome, in v-src-induced cell death

Fig. 2. Effect of cell density on the viability of QNR-ts68 cells. Cells were seeded in 35-mm dishes at increasing cell densities (from left to right: 0.5.105, 105, 0.5.106, 106, 2.106 per dish) and cultured for 18 h under optimal conditions (36.5-C, 10% serum). Cells were then shifted to restrictive temperature (41.5-C) and cultured for 16 more h in presence (41 + S) or absence (41 S) of serum. Apoptosis was evaluated by FACS analysis (fluorescence intensity, Annexin V staining).

in Fig. 5 show that, in QNR-ts68 cells, the inhibition of calpains does not prevent cell shape remodeling due to p60v-src thermal denaturation. To confirm that, in contrast, the proteasome is involved in the observed morphological changes, the effect of CLBL was analyzed. Indeed, this inhibitor provides a rapid means to specifically inactivate the proteasome and is therefore a convenient tool for studying its functions [28]. As shown in Fig. 5A, treatment of QNR-ts68 cells with CLBL, thus inhibiting the proteasome but not calpains (see Figs. 4 and 5B), suppressed cell shape remodeling that followed p60v-src inactivation. Interestingly, the inhibition of protein synthesis with CHX prevented these morphological changes as well, suggesting that cell shape changes depend on protein neosynthesis (Fig. 5A). The effect of protease inhibitors on the cytoskeleton was further analyzed by immunocytochemistry. As shown in Fig. 6, thermal inactivation of p60v-src resulted in the appearance of actin stress fibers, which was correlated with a marked decrease in cell motility and invasiveness (our unpublished results). Moreover, ALLN, but not the three specific calpain inhibitors E64, calpain inhibitor IV and calpeptin, totally inhibited the formation of stress fibers at 41.5-C, confirming that the remodeling of the actin cytoskeleton depends on the ubiquitin –proteasome pathway (Fig. 6). Finally, an interesting observation is that the proteasome is partially down-regulated in QNR-ts68 cells maintained at 41.5-C (Fig. 4A), suggesting that this down-regulation might be necessary for the achievement of cell shape changes after p60v-src inactivation. However, the full inactivation of the

We [13,17] and others [3,29] showed that cells expressing thermosensitive mutants of p60v-src underwent apoptosis following p60v-src inactivation. We previously reported that v-src controlled QNR cell survival via the inhibition of caspase-3 [13] and the activation of nr-13 gene expression [30]. In addition to caspases, a number of proteases are involved in the control of apoptosis. Among them are the proteasome, which activates or inhibits cell death, depending on the cellular microenvironment and/or cell type, and calpains, a family of cysteine proteases that play a key role in cell migration [31]. To investigate which proteases were involved in p60v-src -dependent cell death, QNR-ts68 cells were cultured in the presence of the protease inhibitors ALLN, E64, calpeptin, calpain inhibitor IV, CLBL and Z.VAD. FMK. Cell death was then evaluated by FACS analysis of propidium iodide incorporation. As shown in Fig. 7, micromolar concentrations of ALLN, E64, calpeptin, calpain inhibitor IV or CLBL had

Fig. 3. Time course of caspase-3 activation in QNR-ts68 cells following p60v-src thermal inactivation at restrictive temperature. Whole cell lysates were prepared from QNR-ts68 cells cultured in absence of serum at permissive (36.5-C) or non-permissive (41.5-C) temperature for different durations. Activated caspase-3 was detected by immunoblotting using the CM1 antibody. Anti-tubulin antibody was used for calibration. While serum deprivation for 48 h does not result in the activation of caspase-3 in QNRts68 cells at permissive temperature (left lane, 36.5-C, 48 h), activated caspase-3 is detected in a time-dependent manner after shift up at restrictive temperature (41.5-C, 12 – 48 h).

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Fig. 4. Detection of calpain and proteasome activities in QNR-ts68 cells. Activity assays were carried out on whole cell lysates using the fluorogenic peptide substrates suc-LLVY-MCA and AC-LLY-AFC for 20S proteasome (A) and calpains (B), respectively. Results are expressed as relative fluorescent units per microgram of protein lysate. Lysates were prepared from cells grown at 36.5-C or 41.5-C. Reactions were carried out in absence or presence of indicated inhibitors.

no effect on cell death in QNR-ts68 cells, either at 36.5-C or 41.5-C. In contrast, the pan-caspase inhibitor Z.VAD.FMK prevented cell death at 41.5-C in a dose-dependent manner, having no effect at 36.5-C. Together, these results indicate that, following p60v-src thermal inactivation, QNR-ts68 cells are dying via a caspase-dependent pathway, independently of calpains and the proteasome. Caspases and the proteasome control distinct pathways in v-src-transformed QNR cells Here, we report that the calpain –proteasome inhibitor ALLN, contrary to the caspase inhibitor Z.VAD.FMK, abrogated cellular remodeling following p60v-src inactivation in QNR cells infected with the thermosensitive RSV strain tsNY68. Moreover, we show that the specific proteasome inhibitor CLBL had the same effect on cell shape as ALLN, whereas specific inhibitors of calpains had no effect (Figs. 5 and 6). However, in contrast to Z.VAD.FMK, neither ALLN, calpain inhibitors nor CLBL could maintain cell survival when p60v-src was inactive (Fig. 7). Taken together, these results suggest that the proapoptotic stimulus elicited by p60v-src inactivation recruits at

least the ubiquitin –proteasome pathway for cellular remodeling on the one hand and a caspase-dependent pathway for the execution of cell death on the other hand. In addition, our results indicate that the control of cell shape by p60v-src via the ubiquitin– proteasome pathway is independent of calpains in QNR cells. In contrast, vsrc has been reported to control cell motility and invasiveness via calpain-dependent degradation of the adaptor protein FAK in fibroblasts [3,32]. Thus, v-src seems to control cytoskeletal dynamics via different proteolytic pathways, depending on cell type. This idea is supported by the fact that no degradation of FAK was observed following p60v-src inactivation in QNR-ts68 cells (not shown). Which proteins are degraded following p60v-src inactivation in QNR cells remains to be investigated. In this respect, ezrin, an adaptor protein, which anchors actin fibers to the plasma membrane and is degraded by the proteasome following oxidative stress [33], is an interesting candidate. The cross-talk between the proteasome, caspases and calpains is often complex. Activation of the proteasome may lead to caspase inactivation [34] or activation [35], while calpains can be upstream [36] or downstream [37] of

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Fig. 5. Proteasome inhibitors, but not calpain inhibitors, prevent cell shape changes following p60v-src inactivation. (A) Phase contrast analysis. Cells were grown at permissive temperature (36.5-C) up to the density of 106 cells per 35-mm dish in presence of 10% serum. The cells were then transferred at non-permissive temperature (41.5-C, right panels) with or without indicated inhibitors; cell morphology was observed 16 h after the temperature shift. In control experiments (left panels), cells were maintained for the same duration at 36.5-C. Cell morphology was observed by phase-contrast microscopy. Non-treated cells (MOCK) exhibit marked morphological changes at 41.5-C. In contrast to ZVAD and calpain inhibitors E64, calpain inhibitor IV and calpeptin, the proteasome inhibitors ALLN and CLBL, as well as the inhibitor of protein synthesis CHX prevent these morphological changes. (B) Efficiency of protease inhibitors on calpain activity in vivo. Analysis of calpain activity was performed with the cell-permeable fluorogenic calpain substrate BOC-LM-CMAC in QNR-ts68 cells grown at 36.5-C. In this assay, cells exhibiting calpain activity appear fluorescent (upper left: MOCK; bottom right: CLBL). Calpain inhibitors (ALLN, inhibitor IV, calpeptin), and to a lesser extent E64, significantly decrease fluorescence emission; in contrast, the specific proteasome inhibitor CLBL has no effect.

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Fig. 6. Effect of protease inhibitors on the actin cytoskeleton. Fluorescence analysis. Cells were cultured at 36.5-C or 41.5-C as described above (Fig. 5A), with or without the indicated protease inhibitors. The actin cytoskeleton was subsequently stained with phalloidin-FITC. Right panels: thermal inactivation of the p60v-src tyrosine kinase at 41.5-C results in the appearance of stress fibers, which is prevented by the proteasome inhibitor ALLN, but not the calpain inhibitors E64, calpain inhibitor IV and calpeptin.

caspases, depending on cell type. In QNR cells, we show here that v-src independently controls the activity of the proteasome and caspases. Morphological changes induced by p60v-src inactivation in QNR-ts68 cells are proteasomedependent, while cell death relies mainly on caspases. The exact roles of intracellular proteases in the control of apoptosis, cell shape and adherence, both in normal and cancer cells, remain to be clearly established. They include the identification of key regulatory proteins degraded during these processes. Together, our results should help to improve understanding of the basic mechanisms of neoplastic transformation by the Src family of non-receptor tyrosine kinases.

Materials and methods Cell culture and time-lapse videomicroscopy Neuroretina cells (QNR) were prepared from 7-day-old quail embryos (Coturnix coturnix japonica) and infected with the temperature-sensitive RSV strain tsNY68 (QNRTS68). Cells were grown and passaged in Eagle’s basal medium (EBME, Sigma) supplemented with glutamine, pyruvate and penicillin – streptomycin, in the presence of 10% fetal calf serum (Biowest) as described [13]. Phase contrast image sequences were acquired using an imaging workstation composed of inverted microscope

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Fig. 7. Inhibition of caspases, but not proteasome or calpains, prevents v-src-dependent cell death in ts68-infected QNR cells. Cells (0.5.106 cells per 35 mm dish) were cultured at 36.5-C (vertical gray bars) or 41.5-C (vertical black bars) with 10% serum for 36 h, in the presence or absence of one of the following protease inhibitors: ALLN (10 AM), E64 (10 AM), calpeptin (1 AM), calpain inhibitor IV (5 AM), CLBL (5 AM), Z.VAD.FMK (50 – 200 AM). Cell death was monitored by measuring propidium iodide (PI) incorporation (FACS analysis). The percentage of PI-positive cells is indicated on the vertical axis. Results are means of three independent experiments. Standard error of the mean is shown.

(Axiovert 100 M, Zeiss), temperature and CO2 controlled humidified chamber (37.5-C, 5% CO2) (M incubator, Zeiss), automated shutters (Uniblitz) and CDD camera (Micromax 1300-Y, Princeton-Instruments). The system is coupled with image processing software (Metamorph, Princeton-Instruments), which identifies boundaries from phase contrast images. 100 – 200 cells per field (4 different fields) were scanned every 10 or 30 min for 24 h. Effect of enzyme inhibitors In all experiments, cells were seeded in 35-mm-diameter cell culture dishes in EBME containing 10% serum. After 18 h of culture at 36.5-C, cells were incubated, either at the same temperature or at 41.5-C, with solvent alone (0.2% DMSO, ‘‘mock’’) or with the specified inhibitor for at least 16 more hours in EBME containing 10% serum. For each inhibitor, working concentrations were as follows: Z.VAD.FMK (Bachem, 50 – 200 AM), ALLN (calpain inhibitor I; Calbiochem, 10 AM), E64 (Sigma, 10 AM), calpain inhibitor IV (Calbiochem, 5 AM), calpeptin (Calbiochem, 1 AM), CLBL (Lactacystin; Affinity, 5 AM) and CHX (Sigma, 10 AM). Stock solutions (100 mM) were made in DMSO (Sigma), except for CHX which was resuspended in ethanol.

activated mouse caspase-3 (a gift from A. Srinavasan, Idun pharmaceuticals; see [39]). Calpain activity assays In vitro analysis of calpain activity in total cell lysates was performed using calpain activity assay kit (Biovision) according to manufacturer’s instructions. The calpain activity kit contains a fluorogenic peptide calpain substrate (Ac-LLY-AFC). Briefly, cell lysates were incubated with substrate and reaction buffer for 1 h at 37-C in the dark. Upon cleavage of substrate, the fluorogenic portion (AFC) releases yellow-green fluorescence at 505 nm following excitation at 400 nm. Fluorescence emission was measured by a multiwell standard fluorimeter. Results are expressed as relative fluorescence units per microgram of protein lysate. Analysis of calpain activity in live cells was performed by incubation with the cell-permeable fluorogenic calpain substrate BOC-LC-CMAC (Invitrogen). Substrate contains a fluorogenic subunit and a quencher subunit. Upon cleavage, the fluorogenic portion separates from the quencher, resulting in an increase in fluorescence emission. Cells were incubated with substrate (50 AM) for 20 min. Calpain-mediated cleavage of substrate was visualized by confocal microscopy (magnification 100).

Western blotting and immunodetection of activated caspase-3

In vitro 20S proteasome activity assay

Whole cell lysates were prepared as previously described [17]. Proteins were electrophoresed and transferred onto nitrocellulose membranes using standard protocols (see, for example, [17]). Anti h3-tubulin antibody was used for calibration (see [38]). To detect the activated form of caspase-3, we used affinity-purified rabbit polyclonal antibody raised against a 13-amino-acid-long peptide corresponding to the C-terminus end of the large p20 subunit of

Cells were washed twice with cold PBS and lysed by a 30-min incubation in 0.5 mM dithiothreitol (DTT) as described [40]. Unlysed cells, membranes and nuclei were eliminated by centrifugation at 14,000g. A typical proteasome assay was carried out with 100 Ag of protein cell extract in a total volume of 200 AL of proteasome buffer (50 mM Tris – HCl, pH 7.8, 20 mM KCl, 5 mM MgOAc, 0.5 mM DTT). The fluorogenic substrate suc-LLVY-MCA

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(Sigma) was added to the cell extracts to measure chymotrypsin-like activity. Following incubation at 37-C for 1 h, the reaction was stopped by addition of sodium borate/ethanol (9:1). Fluorescence emission was measured by a multiwell standard fluorimeter. The excitation and emission wave length for aminomethyl coumarine (MCA) were 365 and 460 nm, respectively. Results were expressed as relative fluorescence units per microgram of protein lysate.

[6]

[7] [8]

Immunofluorescence and confocal scanner microscopy [9]

Cells were grown and treated for immunofluorescence detection of the actin cytoskeleton as previously described [17]. FITC-labeled phalloidin was from Molecular Probes.

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Cell viability assays Cells were cultured in the presence or not of the proteases inhibitors as described above. Cell death was routinely monitored using standard protocols (see [41]). For Annexin V labeling, cells were rinsed with PBS to eliminate nonadherent cells and debris and subsequently trypsined, whereas, for propidium iodide incorporation, floating cells and adherent cells were collected. In both cases, cells were then placed in cold PBS for immediate FACS analysis of Annexin V-FITC labeling (according to manufacturer’s instructions, Roche) or for PI incorporation, using FL1 and FL2 channels respectively (FACS Caliburi cytofluorometer) as described [17].

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Acknowledgments [17]

We thank both D. Rigal and J. Bernaud for FACS analyses and A. Srinavasan for the CM1 antibody. This work was supported by grants from the Association pour la Recherche sur le Cancer (ARC), the ‘‘Ligue Nationale Contre le Cancer’’ (comite´ de la Droˆme) and the Re´gion Rhoˆne-Alpes. B.D.N. is supported by a fellowship from the french Ministe`re de la Recherche et de la Technologie, A.L.N. and A.A. are supported by fellowships from the ARC. References [1] G.M. Cohen, Caspases: the executioners of apoptosis, Biochem. J. 326 (Pt. 1) (1997) 1 – 16. [2] L.M. Grimm, B.A. Osborne, Apoptosis and the proteasome, Results Probl. Cell Differ. 23 (1999) 209 – 228. [3] N.O. Carragher, V.J. Fincham, D. Riley, M.C. Frame, Cleavage of focal adhesion kinase by different proteases during SRC-regulated transformation and apoptosis. Distinct roles for calpain and caspases, J. Biol. Chem. 276 (2001) 4270 – 4275. [4] H. Sorimachi, T.C. Saido, K. Suzuki, New era of calpain research. Discovery of tissue-specific calpains, FEBS Lett. 343 (1994) 1 – 5. [5] T.C. Saido, M. Shibata, T. Takenawa, H. Murofushi, K. Suzuki,

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