Translation regulation after taxol treatment in NIH3T3 cells involves the elongation factor (eEF)2

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Translation regulation after taxol treatment in NIH3T3 cells involves the elongation factor (eEF)2 David Piñeiro1 , Víctor M. González1 , Macarena Hernández-Jiménez, Matilde Salinas, M. Elena Martín⁎ Departamento de Bioquímica-Investigación, Hospital Ramón y Cajal, 28034 Madrid, Spain

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

Changes to the translational machinery that occur during apoptosis have been described in

Received 2 March 2007

the last few years. The two principal ways in which translational factors are modified during

Revised version received

apoptosis are: (i) changes in protein phosphorylation and (ii) specific proteolytic cleavages.

24 July 2007

Taxol, a member of a new class of anti-tubulin drugs, is currently used in chemotherapeutic

Accepted 24 July 2007

treatments of different types of cancers. We have previously demonstrated that taxol

Available online 1 August 2007

induces calpain-mediated apoptosis in NIH3T3 cells [Piñeiro et al., Exp. Cell Res., 2007, 313:369–379]. In this study we found that translation was significantly inhibited during

Keywords:

taxol-induced apoptosis in these cells. We have studied the phosphorylation status and

Apoptosis

expression levels of eIF2a, eIF4E, eIF4G and the regulatory protein 4E-BP1, all of which are

eEF2

implicated in translation regulation. We found that taxol treatment did not induce changes

Initiation factors

in eIF2α phosphorylation, but strongly decreased eIF4G, eIF4E and 4E-BP1 expression levels.

Taxol

MDL28170, a specific inhibitor of calpain, prevented reduction of eIF4G, but not of eIF4E or

Translation

4E-BP1 levels. Moreover, the calpain inhibitor did not block taxol-induced translation inhibition. All together these findings demonstrated that none of these factors are responsible for the taxol-induced protein synthesis inhibition. On the contrary, taxol treatment increased elongation factor eEF2 phosphorylation in a calpain-independent manner, supporting a role for eEF2 in taxol-induced translation inhibition. © 2007 Elsevier Inc. All rights reserved.

Introduction Translation is an important target in the regulation of gene expression in response to a large array of extracellular stimuli, playing a key role in controlling cell growth and proliferation. Protein synthesis is divided into three phases: initiation, elongation and termination. Although it is clear that translational regulation is largely exerted at the level of polypeptide

chain initiation, it is becoming evident that regulation at the elongation and termination steps also occurs. The regulation of initiation generally takes place at two stages, the first corresponding to the ternary complex formation between eukaryotic initiation factor 2 (eIF2), GTP and mettRNAi, which takes place prior to the joining of the 40S ribosomal subunit to form the 43S initiation complex; and the second is the recruitment of mRNA to a ribosome, a process that occurs

⁎ Corresponding author. Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal. Ctra. Colmenar km 9,100, 28034 Madrid, Spain. Fax: +34 91 336 9016. E-mail address: [email protected] (M.E. Martín). 1 These authors contributed equally to this work. 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.07.025

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through 5′cap structure recognition (m7GpppX, where “X” is any nucleotide) by eIF4F. In higher eukaryotes, eIF4F consists of three subunits: eIF4E, the cap-binding subunit, eIF4A, an ATPdependent RNA helicase, and eIF4G which serves as a scaffold protein for assembly of eIF4E and eIF4A into the eIF4F complex. The downregulation of translation rates in apoptotic cells was previously reported [1,2], however, only recently the principal changes in the translation machinery that occur during apoptosis have been elucidated. The two principal ways in which translational factors are modified in apoptosis involve (i) changes in protein phosphorylation and (ii) specific proteolytic cleavage (reviewed in [3]). Phosphorylation and dephosphorylation of translation factors play key roles in protein synthesis regulation. Among others, the phosphorylation of the eIF2α subunit converts eIF2 from a substrate to a competitive inhibitor of its exchange factor, eIF2B, and inhibits translation [4]. eIF4E is another factor that can be regulated by phosphorylation although there is conflicting evidence about the role of eIF4E phosphorylation in translational control [5,6]. Under different circumstances, a correlation between increased phosphorylation and enhanced protein synthesis has been observed. However, in other situations, eIF4E phosphorylation has no effect, nor does it even induce a decrease in the rate of total protein synthesis [5]. The effect of eIF4E phosphorylation on eIF4F activity is also controversial; phosphorylated eIF4E was reported to have higher binding affinity for the cap [7] and to form a more stable eIF4F complex [8]. However, another paper reported that eIF4E phosphorylation markedly reduces its affinity for capped mRNA [6]. The specific proteolytic cleavage of eukaryotic initiation factors is another key modification observed under apoptotic conditions. eIF4G (I and II) are proteolytically processed during apoptosis (reviewed in [5] and [9]). In many cases, caspase-3 inhibitors prevent the cleavage of eIF4G, demonstrating that caspase-3 activity is necessary and sufficient for the proteolysis of eIF4G (I and II) both in vivo and in vitro. Other translation factors are substrates for caspase-3, including eIF2α, 4E-BP1, eIF3j and eIF4B [3]. In contrast, there is no evidence of proteolytic cleavage of any elongation factors (eEFs) during apoptosis. During the induction of apoptosis, translation is inhibited with kinetics that varies with the inducing agent and the cell line examined. The mechanism involved in translational regulation also differs. For example, in anti-FAS-treated Jurkat cells, caspase-8 is not required for the increase in eIF2 phosphorylation [10], while in tumor necrosis factor related apoptosis-inducing ligand (TRAIL)treated MCF-7 cells, the phosphorylation of eIF2α is a caspasedependent process, which can be prevented by treatment of the cells with either the broad specificity caspase inhibitor Z-VADfmk or the caspase-8 specific inhibitor Z-IETD-fmk [11]. In another example, cisplatin-induced apoptosis produces cleavage of eIF4GI, eIF4GII and PABP in different cell types, while only PABP is processed in MCF-7 cells [12]. Taxol (paclitaxel) is a potent anticancer agent [13] known to cause polymerization and stabilization of microtubules in tumor cells, thereby inhibiting cell replication through disruption of normal mitotic spindle formation [14,15]. Therefore, cells treated with taxol are unable to proceed normally through the cell cycle and arrest in G2/M phase [16]. This arrest of the cell cycle at mitosis has been considered to be the cause of taxolinduced cytotoxicity [17] and has also been associated with

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apoptosis. Substantial evidence indicates that G2/M arrest is not the only mechanism implicated in taxol-induced apoptosis [17,18]. Thus, additional activities of taxol have also been described including its effect on cell signaling, gene expression, and activation of mitogen-activated protein kinases and Raf-1 [19–21]. Furthermore, taxol triggers apoptosis by both caspasedependent and independent pathways [22–25] that regulate the expression of apoptosis-related proteins like Bim, Bcl-2, Bad, Bcl-XL, p21WAF-1/CIP-1, tumor necrosis factor-α (TNF-α) receptor 1 (TNFR1) and the TRAIL receptors DR4 and DR5 [17,26–30]. In this study we have investigated the translation rate and the changes in the translational machinery that run in parallel with taxol-induced apoptosis in NIH3T3 cells. We found that taxol induced a significant protein synthesis inhibition, due mainly to eEF2 phosphorylation.

Materials and methods Antibodies Rabbit anti-phosphorylated 4E-BP1 (Thr37/46), anti-4E-BP1, anti-caspase-3, anti-phosphorylated eEF2 (Thr 56) and antieEF2 antibodies were purchased from Cell Signaling Technology (Danvers, MA). Monoclonal anti-eIF2α and eIF4E were obtained from AbCam (Cambridge, UK) and BD Biosciences (Franklin Lakes, NJ), respectively. Rabbit anti-phosphorylated eIF2α (Ser51) antibody was purchased from Biosource (Carlsbad, CA). The anti-eIF4G antibody was generously provided by Dr. S.J. Morley. Mouse and rabbit-IgG conjugated peroxidase were purchased from GE Healthcare (Barcelona, Spain) and mouse and rabbit-IgG conjugated alkaline phosphatase were from Sigma (Madrid, Spain).

Cell culture and treatments The mouse fibroblast NIH3T3 cell line was maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum and 100 U/mL penicillin, 100 μg/mL streptomycin and 25 μg/mL amphotericin in a humidified 5% CO2/95% air incubator at 37 °C. Cells were subcultured in 24-well plates at a density of 105 cells/well for examination of viability, protein synthesis rate, and ATP determination, or in 75-cm2 cell culture flasks (1.5 × 106 cells/flask) or 175-cm2 cell culture flasks (2.5 × 106 cells/flask) for all other experiments. After 24 h, cells were pre-treated or not with 50 μM MDL28170 (Calbiochem, San Diego, CA) in DMSO for 1 h and then with 25 μM taxol (Bristol-Myers Squibb, NY) dissolved in 50% EtOH for different time periods (6, 12 and 18 h). In another set of experiments, cells were pre-treated with 20 μM Z-VAD-fmk (Calbiochem) for 1 h before taxol treatment. Control incubations included the same volume of vehicle.

Sample preparation for western blot and isoelectric focusing (IEF) analysis At the end of the treatments the medium was removed and the cells were washed twice with ice-cold buffer A (20 mM Tris– HCl pH 7.6, 1 mM DTT, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine, 2 mM sodium molybdate, 2 mM sodium β-glycerophosphate, 0.2 mM sodium orthovanadate, 120 mM KCl, 1 μg/mL

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leupeptin and pepstatin A, and 10 μg/mL antipain) and then lysed in the same buffer containing 0.5% NP-40 and 0.1% Triton X-100 (50 μL/106 cells). Cell lysates were centrifuged at 12,000×g for 10 min and the supernatants were kept at −80 °C until used. Protein determination was performed by the Bradford method (BioRad, Barcelona, Spain) [31]. Cell lysates were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under the conditions indicated in the figure legends, and transferred onto polyvinylidene fluoride (PVDF) membranes. Monoclonal antibodies were incubated for 1–2 h at room temperature and polyclonal antibodies were incubated at 4 °C overnight. After washing, the membranes were incubated with the corresponding peroxidase-conjugated secondary antibody for 1 h at room temperature, developed with enhanced chemiluminescence kits (GE Healthcare) and exposed to hyperfilm. Full Range Rainbow molecular weight markers (GE Healthcare) were used in all experiments. Fast green FCF (BioRad) staining was used as a control to monitor homogeneity of loading. To determine the eIF4E and eIF2α phosphorylation state, lysates were adjusted to 35 μg of protein per sample, resolved in horizontal isoelectric focusing slab gels (IEF) and analyzed by protein immunoblotting as described previously [32]. Proteins were analyzed by protein immunoblotting with monoclonal antieIF4E and anti-eIF2α antibodies. Alternatively, samples were resolved by SDS-PAGE and analyzed by protein immunoblotting with a phosphospecific antibody against eIF2α (Ser51). Stained bands were scanned and quantified with an analyzer equipped with the Imagequant™ software package (GE Healthcare).

Spain) as described elsewhere [26]. The housekeeping gene GAPDH mRNA was used as a control. 10 and 25 ng of total RNA, obtained from taxol-treated NIH3T3 cells using TRIZOL® Reagent (Invitrogen, Barcelona, Spain), was used as template. The primers used in these reactions were 5′eIF4E (5′-GTCTAGAATGGCGACTGTGGAACCGGA-3′) and 3′eIF4E (5′-GCGGGCCCTTAAACAACAAACCTATTTT-3′) for eIF4E mRNA, and 5′GAPDH (5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′) and 3′GAPDH (5′-CATGTGGGCCATGAGGTCCACCAC-3′) for GAPDH mRNA.

Small interfering RNA (siRNA) design, synthesis and transfection Desalted DNA oligonucleotides were ordered from SigmaGenosys Ltd (Cambridgeshire, UK). Following the procedure described by Elbashir et al. [37], we designed several doublestranded short-interfering RNAs (siRNA) to mouse caspase-3 with 3′ overhanging uridine dimers. Target sequences were aligned to the human genome database in a BLAST search to eliminate those with significant homology to other genes. Four target sequences for the caspase-3 gene were selected for testing. siRNAs corresponding to sequences located in the 5′, 3′, or central regions of the transcript were deliberately chosen

Measurement of protein synthesis rate Protein synthesis rate was assayed in 24-wells plates with fresh medium containing 8 μCi/mL of [3H]Met (GE Healthcare) for 90 min at 37 °C. Cells were harvested, and the incorporation of methionine into protein was determined by trichloroacetic acid precipitation as described previously [32]. In some experiments NIH3T3 cells were incubated with fresh medium, without methionine, containing 10 μCi/well of [35S]Met (GE Healthcare) for 1.5 h before harvesting. Cells were lysed with buffer A as described above, and aliquots containing equal amounts of protein were resolved by SDS-PAGE, stained with Coomassie and exposed to autoradiography.

Degradation of 4E-BP1 by calpain in vitro Five micrograms of recombinant 4E-BP1 (Stratagene, La Jolla, CA) was incubated with 2.4 U/mL of recombinant calpain I (Sigma, Madrid, Spain) and 2 mM CaCl2 at 30 °C in the presence or absence of 50 μM MDL28170. Enzymatic reactions were stopped by mixing 15 μL of each sample with 3× Laemmli buffer, and then subjected to SDS-PAGE (15% polyacrylamide gel) and stained with Coomassie. As control, 5 μg of bovine serum albumin (BSA) or myelin basic protein (MBP) was used as substrate.

Reverse transcription-polymerase chain reaction (RT-PCR) expression analysis eIF4E mRNA expression was assayed by RT-PCR using the Access RT-PCR System (Promega Biotech Ibérica, SL, Madrid,

Fig. 1 – Effect of taxol on protein synthesis in NIH3T3 cells. Cells were treated with taxol for different times and the translation rate was analyzed by [35S]Met incorporation and autoradiography (A) or trichloroacetic acid precipitation and measuring the incorporation of [3H]methionine into protein (B), as described in Materials and methods. (A) Aliquots containing equal amount of labelled protein (12 μg) were resolved by 12% SDS-PAGE, stained with Coomassie (left panel), and the resulting autoradiography is presented in the right panel. (B) Results are expressed as the percentage of control values and represent the mean ± S.E.M. of 3 different experiments. Statistical significance * p < 0.05; ** p < 0.001; *** p < 0.0001. Keys: C, control cells; T, taxol-treated cells.

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to assess whether different regions were more or less susceptible to siRNA-induced degradation. siRNAs were prepared by in vitro transcription using the Ambion Silencer™ siRNA Construction Kit (Austin, TX) and quantified by standard techniques. To suppress caspase-3 expression, NIH3T3 cells were transfected with siRNAs 48 h after plating, using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) follow-

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ing the supplier's instructions. 100 pmol/well of siRNA in 6well plates was used.

ATP determination The amount of cellular ATP was measured with a luminescence-based assay kit (Sigma). Fresh cells were washed with

Fig. 2 – Effect of taxol treatment on phosphorylation state and levels of the translation initiation factors eIF4E and eIF2α. Total lysates from NIH3T3 cells treated with 25 μM taxol for the times shown were subjected to IEF electrophoresis and bands corresponding to eIF4E and eIF4EP (A), and eIF2α and eIF2αP (B) were analyzed by protein immunoblot as described in Materials and methods. Results in figures A and B are expressed as the percentage of eIF4E or eIF2α phosphorylated over total eIF4E or eIF2α, respectively. In another set of experiments, total lysates were resolved by 15% SDS-PAGE, transferred onto PVDF membranes and immunoblotted with anti-eIF4E (C) or anti-eIF2α (D) antibodies. Results in figures C and D are expressed as the percentage of total eIF4E or eIF2α in taxol-treated cells relative to the control. A representative blot is shown at the top of the figures and the membranes were stained with fast green as a loading control. Data represent the mean ± S.E.M. of 3–7 different experiments. Statistical significance * p < 0.05; ** p < 0.001; *** p < 0.0001. Keys: C, control cells; T, taxol-treated cells.

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0.25 mL of cold buffer consisting of 0.1 M Na2HPO4 pH 7.5, and 5 mM EDTA, centrifuged at 1500×g for 5 min, resuspended in 0.2 mL of the same buffer and lysed by sonication (4 × 10 s). After centrifugation at 10,000×g over 15 min, the supernatant (6–8 μg protein) was immediately used for ATP determination. Total ATP was calculated from a standard curve prepared with known amounts of ATP as nmol per mg of protein, and expressed as percentage relative to the control.

and 4E-BP1 levels in the complex were detected by immunoblot analysis and quantified as described above for the individual factors.

Results Protein synthesis inhibition during taxol-induced apoptosis in NIH3T3 cells

Measurement of 4E-BP1/eIF4E and eIF4G/eIF4E complexes The amount of 4E-BP1 and eIF4G recovered when eIF4E was purified using m7GTP-sepharose was carried out as described previously [32]. Protein lysate (75 μg) was incubated with 30 μL of m7GTP-sepharose for 30 min at 4 °C, and washed with buffer A. Proteins were eluted with SDS sample buffer and then subjected to electrophoresis; for eIF4G quantification 7.5% polyacrylamide gel was used, and for both eIF4E and 4E-BP1 quantification 15% polyacrylamide gel was used. eIF4G, eIF4E

We have previously established that 25 μM taxol is capable of inducing apoptosis in 50% of NIH3T3 cells after 18 h of treatment [24]. Herein, we firstly examined the effect of this concentration of taxol on the rate of protein synthesis. As shown in Fig. 1A, taxol induced a significant inhibition of protein synthesis at all the times tested. The quantity of [3H] Met incorporated into proteins demonstrated that translation was inhibited by approximately 50% after 6 h of treatment, reaching a plateau after 12 h (75% inhibition) (Fig. 1B).

Fig. 3 – Effect of taxol treatment on eIF4G and 4E-BP1 levels. Total lysates from NIH3T3 cells treated with 25 μM taxol for the times shown were resolved by 7.5% or 15% SDS-PAGE, transferred onto PVDF membranes and immunoblotted with anti-eIF4G (A), anti-4E-BP1 (B) or anti-phosphospecific 4E-BP1 (Thr37/46) antibodies and reprobed with anti-4E-BP1 antibodies (C). Results in figures A and B are expressed as the percentage of eIF4G or 4E-BP1 in taxol-treated cells relative to the control. A representative blot is shown at the top of the figures and the membranes were stained with fast green as a loading control. Data represent the mean ± S.E.M. of 3–7 different experiments. Statistical significance * p < 0.05; ** p < 0.001.

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Taxol treatment results in a decrease in eIF4G, eIF4E and 4E-BP1 levels, and an increase in the phosphorylation of eIF4E Next, we measured the phosphorylation status and levels of several initiation factors in taxol-treated cells. As shown in Fig. 2, phosphorylated eIF2α and eIF4E levels were analyzed by horizontal isoelectrofocusing (IEF) and western blot analysis. The results obtained indicated that while phosphorylated eIF4E levels were significantly increased after 12 h of treatment (Fig. 2A), no changes were observed in phosphorylated eIF2α levels at any of the times studied (Fig. 2B, Upper panel). This result was confirmed by using a phosphospecific antibody against Ser51 of eIF2α (Fig. 2B, Lower panel). With respect to the expression levels of these two factors, eIF4E levels decreased significantly following 12 h of taxol treatment (Fig. 2C), whereas no change was observed in eIF2α levels (Fig. 2D). We also examined eIF4G levels in taxol treated cells. As shown in Fig. 3A, eIF4G levels decreased significantly in a time dependent manner. We also measured the phosphorylation status and expression levels of 4E-BP1 by its change in migration on a SDS-PAGE gel as detected by western blot analysis. Extracts of control NIH3T3 cells contained approximately equal amounts of 4E-BP1β and the hypophosphorylated α isoforms, while the phosphorylated γ isoform was undetectable. As shown in Fig. 3B, total 4E-BP1 levels decreased abruptly by up to 70% of control after 18 h of treatment. Interestingly, at this time, the 4E-BP1β isoform was the most clearly detected band suggesting an increase in 4E-BP1 phosphorylation. To check this possibility, we have analyzed the phosphorylated residues of 4E-BP1 with a phosphospecific antibody generated against Thr 37/Thr 46. As shown in Fig. 3C, the level of phosphorylated 4E-BP1 diminishes in parallel to the total protein indicating that taxol did not produce any change in 4E-BP1 phosphorylation.

4E-BP1 is a substrate for calpain in vitro Earlier studies have described that both eIF4E and eIF4G proteins are substrates of calpain in vitro [33,34]. Since we have previously demonstrated that taxol induces calpain activation in NIH3T3 cells [24], this activation could account for the reduction of the levels of both factors after taxol-treatment. We have assayed whether or not calpain is also able to cleave 4E-BP1. As shown in Fig. 4, calpain quickly induced degradation of 4E-BP1, and its own lysis (Fig. 4, asterisk). After only 2.5 min, 50% of recombinant 4E-BP1 was processed into two fragments of 16.8 and 14.7 kDa, and after 10 min almost all of 4E-BP1 had been degraded. Other proteins such as BSA (Fig. 4, upper panel) or myelin basic protein (data not shown) were not processed by recombinant calpain. When the incubation was done in the presence of MDL28170, a specific inhibitor of calpain, the degradation of 4E-BP1 was completely blocked indicating that 4E-BP1 was a substrate of calpain in vitro (Fig. 4, lower panel). Thus, calpain could be responsible for the taxolinduced decrease in the translation factor levels.

MDL28170 treatment prevents the taxol-induced decrease of eIF4G, but not of eIF4E and 4E-BP1 In order to address whether or not calpain was responsible for the decrease in the expression of eIF4E, eIF4G and 4E-BP1,

Fig. 4 – 4E-BP1 is a substrate for calpain in vitro. Recombinant 4E-BP1 protein was incubated with calpain I in the presence (lower panel) or absence (middle panel) of MDL28170 for the times indicated in the figure. As a control, BSA was incubated with recombinant calpain as well (upper panel). Samples were resolved by 15% SDS-PAGE and stained with Coomassie. A representative gel of three independent assays is shown. The calculated molecular weight of the two specific cleavage fragments is indicated with a dotted arrow. The asterisk indicates the band corresponding to recombinant calpain.

calpain activity was abolished using 50 μM MDL28170. As shown in Fig. 5, pre-treatment with the calpain inhibitor did not induce any change in eIF4E (Fig. 5A) or 4E-BP1 (Fig. 5B) levels; however, MDL28170 treatment significantly increased the level of eIF4G (Fig. 5C).

The decrease in the amount of eIF4E is due to decreased mRNA levels The above findings show that although eIF4E, 4E-BP1 and eIF4G are substrates for calpain in vitro, only the latter seems to be degraded by calpain after taxol treatment. We decided to use RT-PCR to analyze whether or not 4E-BP1 and eIF4E mRNA levels were affected after taxol treatment. As shown in Fig. 6, there was a strong decrease of eIF4E mRNA in taxol-treated cells for each of the template amounts used in the assay. However, no differences in 4E-BP1 mRNA levels between untreated and taxol-treated cells were observed (data not shown), suggesting that the decrease in 4E-BP1 levels was not a consequence of mRNA degradation. This result supports the hypothesis that the decrease in eIF4E protein levels induced by taxol was a consequence of decreased mRNA level.

Caspase-3 is not responsible for the reduction of 4E-BP1 observed in MDL28170 pre-treated taxol-treated cells We have previously reported that the calpain inhibitor MDL28170 produces higher caspase-3-dependent apoptosis in taxol-treated cells [24]. In addition, caspase-mediated cleavage of 4E-BP1 has been shown to occur in cells exposed to staurosporine and the DNA-damaging agent etoposide [35,36]. It is possible that caspase-3, which is activated in the presence of taxol and MDL28170, can cleave 4E-BP1, thereby avoiding the recuperation of its levels under these conditions. In order to test this possibility, we generated four siRNAs against mouse caspase-3 by targeting different positions

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Fig. 5 – Effect of MDL28170 on eIF4E, 4E-BP1 and eIF4G levels. NIH3T3 cells were pre-incubated with or without 50 μM MDL28170 for 1 h and then treated with 25 μM taxol for 18 h. Total lysates, prepared as described in Materials and methods, were resolved by 15% or 7.5% SDS-PAGE, transferred onto PVDF membranes and immunoblotted with anti-eIF4E (A), anti-4E-BP1 (B) or anti-eIF4G (C) antibodies. Results in the figures are expressed as the percentage of eIF4E, 4E-BP1 or eIF4G in treated cells relative to the control. A representative blot is shown at the top of the figures and the membranes were stained with fast green as a loading control. Data represents the mean ± S.E.M. of 3–7 different experiments. Statistical significance with respect to control cells * p < 0.05; ** p < 0.001; *** p < 0.0001. Statistical significance respect to taxol-treated cells c p < 0.05.

within the coding region of caspase-3 according to the procedure described by Elbashir et al. [37]. As a negative control, a non-specific siRNA (NS) was also designed. To examine the ability of the siRNAs to suppress endogenous caspase-3 expression, NIH3T3 cells were transfected with the siRNAs and harvested 24, 48 and 72 h post transfection, and the level of caspase-3 was determined by western blot analysis (Fig. 7A). Caspase-3 expression was strongly decreased in cells transfected with all of the siRNAs 48 h after transfection, diminishing by more than 50% after 72 h. siRNA-C47 was subsequently chosen for this study. Next, we studied the effect of the reduction of caspase-3 expression on 4E-BP1 levels. As shown in Fig. 7B, down

regulation of caspase-3 by siRNA did not induce any change in 4E-BP1 levels in the control cells. As described above, in cells treated with taxol, regardless of pre-treatment with MDL28170, there was a significant reduction of 4E-BP1 that was not prevented by caspase-3 siRNA transfection. We also included in the analysis of 4E-BP1 cell extracts treated with 20 μM ALLN because, as we showed in a previous paper [24], ALLN induces caspase-3 activation in NIH3T3 cells. The results showed that the antibody directed against 4E-BP1 used in this study was able to detect the caspase-mediated cleavage product of 4E-BP1 (Fig. 7B, arrowhead). In addition, we have also used the caspase inhibitor Z-VAD-fmk in the presence or the absence of taxol and MDL28170. As shown in Fig. 7C, the

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caspase-3 was not implicated in the reduction of 4E-BP1 levels observed in MDL28170 pre-treated taxol-treated cells.

Protein synthesis inhibition is neither due to ATP depletion nor decreased levels of the eIF4E complex

Fig. 6 – RT-PCR analysis of the expression pattern of eIF4E. NIH3T3 cells were treated with or without 25 μM taxol for 18 h and total RNA was prepared as described in Materials and methods. Several concentrations of total RNA were used in a RT-PCR assay. The experiment shown is representative of several independent experiments. Keys: −, negative control (reaction without RNA template). The housekeeping gene GAPDH mRNA was included as a control.

inhibition of caspase-3 activity was not sufficient to abrogate 4E-BP1 decrease in taxol-treated cells pre-treated with MDL28170. All these results support the hypothesis that

To test whether or not the taxol-induced eIF4G depletion is involved in the decrease of protein synthesis, we measured the protein synthesis rate in both the absence and presence of MDL28170 (Fig. 8A). Our results showed that MDL28170 was unable to prevent taxol-induced translation inhibition; in fact MDL28170 produced an additional 20% decrease in the translation rate. Protein synthesis is a process that consumes a considerable amount of ATP. Because of this, it seemed interesting to us to measure the ATP concentration in taxol-treated cells that were pre-treated or not with MDL28170. As seen in Fig. 8B, MDL28170-pre-treated taxol-treated cells showed 3-fold higher amounts of ATP in comparison to non-pre-treated taxol-treated cells, and an ATP concentration 50% higher than control cells. This fact suggested that factors other than ATP depletion were responsible for translation inhibition.

Fig. 7 – Effect of caspase-3 suppression by siRNA on 4E-BP1 levels. (A) NIH3T3 were transfected with several siRNAs as described in Materials and methods, and total lysates (60 μg) were resolved by 12% SDS-PAGE, transferred onto PVDF membranes and immunoblotted with anti-caspase-3 antibody. (B) Caspase-3 and 4E-BP1 expression in NIH3T3 cells pre-incubated or not with 50 μM MDL28170 for 1 h and treated or not with 25 μM taxol for 18 h, and transfected with siRNA-NS or siRNA-C47 were determined as in (A) by 15% SDS-PAGE. In the last lane, we included the detection of 4E-BP1 in lysates from cells treated with 20 μM ALLN. The caspase-mediated cleavage product of 4E-BP1 is indicated with an arrowhead. (C) NIH3T3 cells were pre-incubated or not with 50 μM MDL28170 and/or 20 μM Z-VAD-fmk for 1 h and then treated or not with 25 μM taxol for 18 h. 4E-BP1 expression was determined as in (B). A representative blot from 2 to 3 independent experiments is shown. As a control for loading, the membranes were stained with fast green. Keys: NS, non-specific siRNA; C18, C44, C47 and C88 indicate the four siRNA obtained for caspase-3.

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To examine eIF4F activity, lysates from taxol-treated cells pre-treated with or without MDL28170 were subjected to affinity chromatography on m7GTP-sepharose and the bound proteins were analyzed by western blot analysis. As shown in Fig. 8C, eIF4F complex formation, determined by the associ-

ation of eIF4G and eIF4E bound to m7GTP-sepharose, was 56 ± 12% in taxol-treated cells relative to control cells. As expected, in cells pre-treated with the calpain inhibitor before taxol treatment the eIF4F complex formation was higher (73.9 ± 10.2%), while 4E-BP1 bound to eIF4E decreased both in taxol-

Fig. 8 – Effect of ATP, eIF4F and eIF4E:4E-BP1 complexes on protein synthesis inhibition during taxol-induced apoptosis. (A) NIH3T3 cells pre-treated or not with MDL28170 were treated with taxol for 18 h and the translation rate was analyzed by trichloroacetic acid precipitation and measuring the incorporation of [3H]methionine into protein as described in Fig. 1. Values are expressed as percentage relative to the control. (B) NIH3T3 cells were treated as above and the amount of cellular ATP was measured as indicated in Materials and methods. Values are expressed as percentage relative to the control. (C) eIF4E and its associated proteins were isolated by m7GTP-sepharose affinity chromatography from extracts of cells treated as described in Materials and methods. The recovered proteins were resolved by SDS-PAGE, transferred onto PVDF membranes and immunoblotted with eIF4G, eIF4E and 4E-BP1 antibodies. The relative levels of the factors were quantified by scanning densitometry and are expressed as the percentage of eIF4G and 4E-BP1 associated with eIF4E in treated cells relative to control cells. A representative blot is shown at the top of the figure. Data represents the mean ± S.E.M. of 3–5 different experiments. Statistical significance with respect to control cells * p < 0.05; ** p < 0.001; *** p < 0.0001. Statistical significance respect to taxol-treated cells c p < 0.05; b p < 0.01.

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treated cells (41.2 ± 7%) and in MDL28170-pre-treated taxoltreated cells (50.5 ± 12.6%) in a similar way (Fig. 8C, lower panel). Surprisingly, a clear decrease in the amount of eIF4F complex was also observed in MDL28170-pre-treated cells (61.1 ± 10.2%) without a change in the amount of 4E-BP1 bound to eIF4E (106.6 ± 5.6%). Therefore, these results support the lack of a role for 4E-BP1 in taxol-induced inhibition of protein synthesis.

eEF2 regulates translation in taxol-induced apoptosis Another factor that can also regulate the protein synthesis rate is the elongation factor 2 (eEF2). An important role in the regulation of translation is generally assigned to the phosphorylation of eEF2 at Thr56, which inhibits the eEF2 activity. Several pathways seem to be regulating eEF2 phosphorylation. One of them is mediated by the cytosolic calcium increase that activates a specific Ca2+ /calmodulin-dependent kinase, termed eEF2 kinase (eEF2K) [38]. Since we have previously demonstrated that taxol induces a significant increase in cytoplasmic Ca2+ concentration in NIH3T3 cells [24], we decided to study the phosphorylation status and levels of eEF2. As shown in Fig. 9A, eEF2 phosphorylation is significantly increased in NIH3T3 cells, reaching an increase of 4.86fold after 6 h of treatment which is maintained along the time. Moreover, a decrease in eEF2 levels by up to 40% can be observed after 6 h of treatment. Finally, we observed that MDL28170 completely prevented taxol-induced eEF2 degradation without affecting its phosphorylation status (Fig. 9B).

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Discussion In this study, we described the suppression of protein synthesis during the taxol-induced apoptosis in NIH3T3 cells for first time, and have thoroughly examined the mechanisms underlying this process. It has been reported previously that global protein synthesis is strongly inhibited during apoptosis induced by different stimuli [1–3,39]. With regard to the first step of initiation regulation (i.e., eIF2B inhibition by eIF2 phosphorylation), we did not observe a significant change in the phosphorylation of eIF2α during taxol-induced apoptosis. While initially surprising, given reports showing eIF2α phosphorylation during apoptosis in response to different inducers [10,11], our results are consistent with others using apoptotic stimuli such as activation of p53 protein [40] or etoposide [35]. Another controversial new result revealed by this work is the relative increase in eIF4E phosphorylation. In fact, a decrease in the phosphorylation state of eIF4E during apoptosis has been described, which may be a consequence of caspase-3-mediated eIF4G cleavage, because the binding site of the eIF4E kinase Mnk1 is not present in the Fragments of Apoptotic cleavage of eIF4G (M-FAG) that bind eIF4E [9,41]. However, other authors have found eIF4E phosphorylation in etoposide-induced apoptosis even in the presence of caspase3-mediated eIF4G cleavage [39]. Although eIF4E phosphorylation is mainly associated with an increase in protein synthesis, the role of this phosphorylation in translational regulation remains to be elucidated.

Fig. 9 – Effect of taxol and MDL28170 on phosphorylation state and levels of eEF2. (A) Total lysates from NIH3T3 cells treated with 25 μM taxol for the times shown were subjected to 10% SDS-PAGE, transferred onto PVDF membranes and immunoblotted with anti-phosphorylated eEF2 (Thr56) polyclonal antibody and then reprobed with the corresponding anti-eEF2 antibody as described in Materials and methods. (B) Total lysates from NIH3T3 cells pre-treated or not with MDL28170 and then treated with taxol for 18 h were resolved on a 10% SDS-PAGE gel, transferred onto PVDF membranes and immunoblotted as in (A). Bars represent the percentage of total eEF2 relative to the control. A representative blot is shown at the top of the figure. Numbers at the bottom of the blots represent the ratio, calculated from 3 to 4 different experiments, between phosphorylated and total eEF2 relative to the control that is considered as 1. Statistical significance with respect to control cells * p < 0.05; ** p < 0.001.

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Several alterations in the translation initiation factors involved in the regulation of the eIF4F complex were observed during taxol-induced apoptosis. A significant decrease in the levels of eIF4E, eIF4G and the regulatory protein 4E-BP1 were observed after taxol treatment, but these changes were regulated in different ways. Firstly, since the levels of eIF4G were recovered after pre-treatment with the calpain inhibitor MDL28170, this clearly indicated that this factor was degraded by calpain activation promoted by taxol-treatment [24]. Calpain was previously shown to become activated during thymocyte apoptosis [42] and ischemic injury when calpain-mediated eIF4G degradation has been also reported [33,43–46]. Other studies have shown that eIF4E is also a substrate of calpain [34]. Nevertheless, in our model, the degradation of eIF4E was not inhibited by MDL28170 pre-treatment, which suggested that the factor is not a substrate of calpain in vivo. In fact, our findings demonstrated that the reduction in the quantity of eIF4E observed after taxol treatment is a consequence of decreased eIF4E mRNA levels. This decrease is probably due to apoptosis-mediated mRNA degradation, as described in a number of different studies [47–49]. Neither of these mechanisms, mRNA diminution or calpain activation, seemed to be regulating the decrease in 4E-BP1 levels in taxol-treated cells. In this paper, we have also demonstrated for the first time that 4E-BP1 is a substrate for calpain in vitro. However, since MDL28170 pre-treatment did not affect 4E-BP1 cleavage, we can conclude that, at least under our conditions, 4E-BP1 is not a substrate for calpain in vivo. To discard the participation of MDL28170-induced caspase-3 activation in the 4E-BP1 cleavage, we used siRNAs against caspase-3 or the caspase inhibitor zVAD-fmk to inhibit its expression or activity, respectively. These approaches revealed that caspase-3 is not implicated in the reduction of 4E-BP1 levels observed in MDL28170 pre-treated taxol-treated cells. It has been reported that the levels of 4E-BP1 decrease strongly under different physiological conditions, such as following fertilization of sea urchin eggs [50] or in squirrels during summer [51]. With regard to our model, a decrease in 4E-BP1 levels would result in increased free eIF4E levels and translation up-regulation instead of the observed translation inhibition. Protein synthesis is one of the most energy-dependent biological processes and consequently consumes a high quantity of ATP. When we investigated the concentration of ATP in taxol-treated cells, a strong depletion was observed after 18 h of treatment, suggesting that low quantities of ATP could be responsible for the inhibition of protein synthesis. However, after MDL28170 pre-treatment protein synthesis continued to be inhibited, and the levels of ATP remained very high, demonstrating that factors other than ATP depletion have to be implicated in translation inhibition. The association of eIF4G with eIF4E strongly enhances the binding of the latter to 5′ mRNA cap structures. Thus, the decrease observed in both eIF4E and eIF4G levels after taxol treatment suggested that protein synthesis inhibition could be due to a reduction of eIF4F complex formation, mainly through the disruption of cap-initiation complexes. The finding that eIF4F complex formation was reduced by half in taxol-treated cells compared to controls supported the hypothesis that taxolinduced protein synthesis inhibition could be due to the diminished formation of the eIF4F complex. However, in MDL28170-pre-treated cells the eIF4F complex was also dimin-

ished relative to control cells, but protein synthesis was unaffected, clearly suggesting that the amount of eIF4F complex in taxol-treated cells was sufficient to maintain the protein synthesis rate at control levels. In addition, the amount of the eIF4F complex is similar in MDL28170-pre-treated cells to that in MDL28170-pre-treated taxol-treated cells where protein synthesis rate is even lower than in taxol-treated cell, supporting the notion that the amount of eIF4F complex is not involved in taxol-induced protein synthesis inhibition. In view of the fact that all our findings indicated that none of the initiation factors or ATP depletion seemed to be responsible for the inhibition of protein synthesis observed after taxol treatment, we decided to address whether or not the elongation step was involved in the regulation of the global translation rate in taxol-treated cells. Phosphorylation of eEF2 by eEF2K prevented eEF2 binding to ribosomes, which resulted in a further elongation inhibition. eEF2K is normally dependent on Ca2+/ calmodulin and can be activated by PKA in response to elevated cAMP levels. An increased AMP/ATP ratio by ATP depletion or the direct activation of the AMP-dependent protein kinase (AMPK) by AMP leads to eEF2 phosphorylation, probably through activation of eEF2K. mTOR pathways also potentially modulate eEF2K activity by phosphorylation (reviewed in [38]). On the other hand, eEF2 levels in most cells seem to be approximately stoichiometric with the number of ribosomes. Given this parity, an alteration in eEF2 protein level is likely to influence the overall ratio of translation [52]. A very interesting finding in this paper is that eEF2 is a substrate for calpain in vivo, as demonstrated by the fact that in MDL28170-pre-treated taxol-treated cells eEF2 levels are recovered relative to taxol-treated cells. As for the role of translation elongation in taxol-induced apoptosis, an increase in eEF2 phosphorylation upon treatment of cells was observed. On the basis of the above findings, we propose a model to explain taxol-induced protein inhibition by elongation regulation in which taxol-induced Ca+2 elevation [24] activates eEF2K and calpain, inducing an increase in phosphorylated eEF2 levels and eEF2 degradation, respectively. These changes would lead to inactivation of eEF2 and elongation inhibition. Treatment with the calpain inhibitor MDL28170 prevented the reduction in eEF2 levels, but not the increase in intracellular Ca+2, and consequently the eEF2K activation and eEF2 inactivation. In summary, we have demonstrated a strong decrease in protein synthesis after taxol treatment that occurs simultaneously with apoptosis in NIH3T3 cells. We have studied the most important initiation factors (eIF2α, eIF4E and eIF4G) and the regulatory protein 4E-BP1, all of which are implicated in translation regulation, and demonstrated that none of them are responsible for the inhibition of protein synthesis. Since translation is one of the most energy-dependent biological processes, we analyzed the ATP levels and demonstrated that it is not limiting for protein synthesis in our model. Finally, our findings strongly point out that protein synthesis inhibition after taxol treatment could be produced at the elongation level due to phosphorylation of the factor eEF2.

Acknowledgments This work was supported by grant FIS03/0469 from Fondo de Investigaciones Sanitarias (FIS) of the Ministerio de Sanidad y

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Consumo (Spain). D. Piñeiro is a fellow from FIS03/0469. Dr. M.E. Martín is a researcher from FIBio-HRC supported by Consejeria de Sanidad y Consumo (CAM) and Dr. V.M. González is a researcher from FIBio-HRC supported by research contract from FIS and Consejeria de Sanidad y Consumo (CAM). We gratefully acknowledge M. Isabel Pérez-Morgado for technical assistance and Dr. Simon J. Morley for kindly providing the anti-eIF4G antibody.

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