p53-induced inhibition of protein synthesis is independent of apoptosis Constantina Constantinou 1 , Martin Bushell 1 *, Ian W. Jeffrey 1 , Vivienne Tilleray 1 , Matthew West 1 , Victoria Frost 2 †, Jack Hensold 3 and Michael J. Clemens 1 1 Translational Control Group, Department of Basic Medical Sciences, St George’s Hospital Medical School, Cranmer Terrace, London; 2 Biochemistry Group, School of Biological Sciences, University of Sussex, Falmer, Brighton, UK; 3 Department of Hematology and Oncology, Case Western Reserve University and the Veterans Administration, Cleveland, Ohio, USA Activation of a temperature-sensitive form of p53 in murine erythroleukaemia cells results in a rapid impairment of protein synthesis that precedes inhibition of cell proliferation and loss of cell viability by several hours. The inhibition of translation is associated with specific cleavages of polypep- tide chain initiation factors eIF4GI and eIF4B, a pheno- menon previously observed in cells induced to undergo apoptosis in response to other stimuli. Although caspase activity is enhanced in the cells in which p53 is activated, both the effects on translation and the cleavages of the initiation factors are completely resistant to inhibition of caspase activity. Moreover, exposure of the cells to a combination of the caspase inhibitor z-VAD.FMK and the survival factor erythropoietin prevents p53-induced cell death but does not reverse the inhibition of protein synthesis. We conclude that the p53-regulated cleavages of eIF4GI and eIF4B, as well as the overall inhibition of protein synthesis, are caspase-inde- pendent events that can be dissociated from the induction of apoptosis per se. Keywords: caspases; erythroleukaemia; p53; protein synthe- sis; temperature-sensitive mutants. The tumour suppressor protein p53 is a key regulator of both cell cycle progression and cell death by apoptosis [1–5]. Inactivating mutations of p53 have been found with high frequency in a broad spectrum of tumours and the inactivation of p53 is central to the transforming function of several viral oncoproteins [6–8]. Primarily, p53 functions as a transcription factor controlling expression of genes that affect cell proliferation, induce DNA repair or regulate cell survival [9–12]. Expression of p53 in p53-negative cell lines induces a cell cycle block and in many cases results in cell death by apoptosis [1,13]. p53 has also been demonstrated to control the activity of RNA polymerases I and III, suggesting that p53 regulates the synthesis of ribosomes and tRNAs [14]. Furthermore, the tumour suppressor protein has been found in association with ribosomes [15,16] and has been shown to have an effect on the translation of specific mRNAs, such as those encoding cdk4, fibroblast growth factor (FGF) 2 and p53 itself [14,17–21]. Recently, we reported that p53 down-regulates overall translation at the level of polypeptide chain initiation [22]. In those studies we utilized a murine erythroleukaemia (MEL) cell line expressing a temperature-sensitive p53 mutant (Val135) [23] and showed that activation of p53 by placing the cells at 32 °C caused a rapid decrease in the overall rate of protein synthesis. However it has not been established whether this translational inhibition is an early part of the programme of induced cell death or whether it is associated with the block to cell cycle progression mediated by the activation of p53. There are strong precedents for the former as several studies have shown that the induction of apoptosis by other agents is accompanied by a substantial down-regulation of translation and the caspase-mediated cleavage of certain polypeptide chain initiation factors [24–29]. In the work described here we have employed the same MEL cell system to address some of these issues. Careful comparisons of the kinetics of translational down-regula- tion vs. the inhibition of cell cycle progression and induction of apoptosis show that the effect of p53 activation on protein synthesis is an early event that precedes both overt inhibition of cell proliferation and the loss of cell viability. We show that although caspase Correspondence to M. J. Clemens, Department of Basic Medical Sciences, St George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK. Fax: + 44 (0)20 87252992, Tel.: + 44 (0)20 8725 5762, E-mail: M.Clemens@sghms.ac.uk Abbreviations: 4E-BP1, eIF4E binding protein 1; Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp7-amino-4-methylcoumarin; Ac-IETD- AMC, acetyl-Ile-Glu-Thr-Asp7-amino-4-methylcoumarin; Ac-LEHD-AMC, acetyl-Leu-Glu-His-Asp7-amino-4-methylcou- marin; eIF, eukaryotic initiation factor; Epo, erythropoietin; MEL, murine erythroleukaemia; mTOR, mammalian target of rapamycin; PARP, poly(ADP-ribose) polymerase; RFU, relative fluorescence units; TNF-a, tumour necrosis factor a; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand; z-VAD.FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. *Present address: Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK. Present address: School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK. (Received 11 March 2003, revised 21 May 2003, accepted 27 May 2003) Eur. J. Biochem. 270, 3122–3132 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03687.x activities increase within a few hours of activating p53, and specific proteolytic cleavages of some polypeptide chain initiation factors are observed, the factor cleavages do not depend on caspase activity. Clonogenicity assays have established that the cells do not become irreversibly committed to apoptosis until several hours after the initial inhibition of translation. Moreover, conditions that block apoptosis do not prevent the p53-induced translational down-regulation. Our results are consistent with a mech- anism whereby the p53-mediated inhibition of protein synthesis in MEL cells is at least partially mediated by initiation factor cleavages. However caspase activity is not required for these cleavages and the down-regulation of translation can be dissociated from the p53-induced apoptotic programme. Materials and methods Cell culture conditions The Val135 and Pro190 MEL cell lines were obtained from S. Benchimol [30] and were grown in stationary suspension culture in DMEM medium supplemented with glutamine (300 mgÆL )1 ) and 10% (v/v) fetal bovine serum in a 5% CO 2 atmosphere at 38 °C. Under these conditions the cells had a doubling time of approximately 12 h. Cultures were maintained at densities between 2 and 8 · 10 5 cells per milliliter. Continued expression of p53 was assured by weekly selection of the cells in G418 (200 lgÆmL )1 ). For activation of p53 in the Val135 cells the cultures were transferred to 32 °C for the times indicated. The control Pro190 cells were treated similarly. Where indicated, the cells were treated with erythropoietin (Epo) and caspase inhibitors at the concentrations described in Table 2 and legends to Figs 4–6. Analysis of cell proliferation, the cell cycle and clonal growth potential Cells were counted in triplicate using a haemocytometer and cell viability was determined by trypan blue exclusion. For cell cycle analysis cells were examined by flow cytometry as described in [31]. Cells that had been grown at 38 °Cor incubated at 32 °C for various periods of time (10 7 cells per sample) were centrifuged at 1000 g for 5 min and washed three times in 5 mL NaCl/P i . The pellets were resuspended in approximately 500 lLofNaCl/P i , 5 mL of cold ethanol were added and the cells were fixed at 4 °Covernight. The fixed cells were washed in NaCl/P i and stained with propidium iodide (500 lgÆmL )1 ). After treatment with boiled RNAse A the cells were analyzed on a FACS flow cytometer (Beckton Dickinson). To determine the ability of the cells to proliferate clonally after exposure to the p53 permissive temperature, Pro190 and Val135 cells were diluted 1 · 10 5 such that the final concentration was three cells per milliliter. Aliquots of 100 lL were added to the wells of 96-well microtiter plates (giving an average of one cell for every three wells). The plates were incubated for various times up to 72 h at 32 °C andthenreturnedto38°C. The wells were observed microscopically after a total of 10 days and were scored for the number of clones that had proliferated. Measurement of protein synthesis rates Overall rates of protein synthesis were measured by pulse- labeling intact cells for up to 1 h with 10–15 lCiÆmL )1 of [ 35 S]methionine (in the presence of the normal level of methionine in the cell culture medium). The cells were centrifuged briefly at 1000 g, washed once in cold NaCl/P i , dissolved in 0.3 M NaOH and precipitated with 10% trichloroacetic acid in the presence of 0.5 mg bovine serum albumin carrier protein. Precipitates were harvested on GF/ C filters under suction and washed with 5% trichloroacetic acid and industrial methylated spirit. The acid-insoluble radioactivity was determined by scintillation counting. Preparation of cell extracts and analysis by immunoblotting Cytoplasmic extracts were prepared for immunoblotting by washing the cells in NaCl/P i and lyzing them in a buffer containing a cocktail of protease and protein phosphatase inhibitors [24]. The extracts were analyzed by SDS gel electrophoresis using equal amounts of protein in each lane of the gel (3–10 lg protein per sample). After transfer of the proteins to poly(vinylidene difluoride) membranes the blots were blocked and incubated with the appropriate primary antibodies against polypeptide chain initiation factors eIF4B and eIF4GI. The blots were developed with alkaline phosphatase-linked secondary antibodies using nitroblue tetrazolium as the substrate [24], or with horseradish peroxidase-linked secondary antibodies followed by enhanced chemiluminescence. As a positive control for the effects of z.VAD-FMK, the same initiation factors were also examined in extracts from Jurkat cells treated with an agonistic anti-Fas (CD95) antibody, as described previously [32,33]. Measurements of apoptosis The progress of apoptosis in the MEL cells was assessed by measuring the activities of caspases-3, -8 and -9 in cell extracts. At appropriate times after incubation at 32 °C, in the absence or presence of z.VAD-FMK, aliquots of 10 7 cells were washed with NaCl/P i , resuspended in 1 mL cell lysis buffer (10 m M Hepes, pH 7.3, 2 m M EDTA, 0.1% NP-40, 5 m M dithiothreitol, 1 m M phenylmethanesulfonyl fluoride, 10 lgÆmL )1 pepstatin A, 20 lgÆmL )1 leupeptin, 10 lgÆmL )1 aprotinin) and incubated on ice for 10–15 min. After centrifugation of the extracts at 10 000 g for 1 min at 4 °C the supernatants were frozen at )80 °C. Caspase activities using fluorogenic substrates were determined in a Packard Fusion microplate reader. Twenty microliters of each cell extract was incubated with 200 lL of reaction buffer [100 m M Hepes, pH 7.3, 20% (v/v) glycerol, 0.5 m M EDTA, 5 m M dithiothreitol] and 2 lL of substrate for caspases-3, -8 or -9 (Ac-DEVD-AMC, Ac-IETD-AMC or Ac-LEHD-AMC, respectively) (Biosource International), each at 5 m M . Reactions were incubated at 37 °Cfor1h and the product was quantified by fluorescence using an excitation wavelength of 380 nm and an emission wave- length of 460 nm. Protein concentrations were determined and caspase activities expressed in relative fluorescence units (RFU) per microgram of protein. Apoptosis was Ó FEBS 2003 Regulation of protein synthesis by p53 (Eur. J. Biochem. 270) 3123 also assessed by the cleavage of the caspase substrates poly(ADP-ribose) polymerase (PARP) and p27 KIP1 ,using immunoblotting procedures as described elsewhere [24,34,35]. Results Inhibition of protein synthesis and cell proliferation following activation of p53 Activation of the temperature-sensitive Val135 p53 mutant in MEL cells (or of the equivalent Val138 mutant in human cells) can be achieved by reducing the incubation tempera- ture from 38 to 32 °C and results in inhibition of cell proliferation and subsequent induction of apoptosis [36–39]. Figure 1A shows the kinetics of cell growth at the two temperatures of the Val135 cells, containing the tempera- ture-sensitive p53, in comparison with that of Pro190 cells which express a mutant form of p53 that is inactive at either temperature. Both cell lines grew at approximately equal rates at 38 °C, with a doubling time of about 12 h. At 32 °C the growth rates were slower but again approximately the same for the first 24 h, during which both cell lines completed at least one traverse of the cell cycle. After this time, whereas the Pro190 cells continued to proliferate, the Val135 cells showed no further increase in number and indeed exhibited a decline over the ensuing 24–48 h. Cell cycle analysis of the Val135 cells (Fig. 1B) indicates that after about 24 h at 32 °C there was a substantial decrease in the fraction of cells in G2/M relative to G1, consistent with a cell cycle block in the G1 phase. At this time very few cells showed a sub-G1 DNA content, in contrast to the situation at later times (Fig. 1B), suggesting that overt apoptosis does not begin until after 24 h. Pro190 cells showed neither any significant shift in cell cycle distribution nor any evidence of apoptosis, even after 72 h at 32 °C (data not shown). Consistent with the cell cycle analysis, the viability of the Val135 cells remained high up to 20 h at 32 °C but declined substantially thereafter, as judged by trypan blue exclusion assays (Fig. 2A). In contrast to the delayed effects of p53 activation on cell proliferation and viability, the shift to the lower temperature resulted in an early inhibition of the overall rate of protein synthesis in the Val135 cells, relative to that in the Pro190 cells (Fig. 2A). Thus comparison of the kinetics of inhibition with the rate of decline in cell viability and the appearance of apoptotic cells shows that the p53-mediated decrease in translational activity preceded cell death by several hours. We also investigated whether the translational inhibition, although preceding overt apoptosis, may nevertheless act as a signal to commit cells to death. To test this, we measured the ability of Val135 cells to recover when replaced at 38 °C after various lengths of exposure to the p53-permissive temperature. Figure 2B shows that the majority of cells retained the ability to survive and recover after incubation at 32 °C for up to 16 h (as judged by their potential for subsequent clonal growth at 38 °C). This was in spite of the fact that the overall rate of protein synthesis progressively declined by up to 50% over this time period. However, after 20 h or more at 32 °C the ability of the cells to recover declined sharply, coinciding with the onset of cell death indicated by the failure to exclude trypan blue. Taken together with the data in Fig. 1 these results suggest that the effect of p53 on protein synthesis cannot merely be a consequence of either the cessation of cell proliferation or the loss of cell viability as it precedes both these events in the temperature-sensitive MEL cells incubated at the permissive temperature. Moreover, translational inhibition per se for up to 16 h is not sufficient to induce cell death. Neverthe- less, as we have not yet identified a means of preventing the down-regulation of protein synthesis, we cannot exclude a requirement for longer periods of inhibition for the p53-mediated induction of subsequent apoptosis. Fig. 1. Inhibition of cell proliferation and changes in cell cycle distri- bution following activation of p53. (A) Exponentially growing Val135 and Pro190 MEL cells were diluted to 1.3 · 10 5 cellsÆmL )1 and incu- bated at 38 °Cor32°C for the times indicated. Total cell numbers were determined in quadruplicate in a haemocytometer. The values shown are means ± SEM. (B) Exponentially growing Val135 MEL cells were maintained at 38 °C or transferred to 32 °Cfor24hor48h. The cells were fixed with ethanol and then stained with propidium iodide as described in Materials and methods. The distribution of the cells in the cell cycle was determined by FACS analysis of DNA content. The peaks corresponding to cells with a sub-G1, G1 or G2/M DNA content are indicated. 3124 C. Constantinou et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Initiation factor cleavages following activation of p53 Previously several changes to the protein synthetic machin- ery have been observed to occur during the early stages of apoptosis in a range of cell types. These include the specific, caspase-dependent cleavage of polypeptide chain initiation factors such as eIF4GI, eIF4B and 4E-BP1 [24–29]. We have therefore examined extracts from Val135 and Pro190 MEL cells for the integrity of eIF4GI and eIF4B following the shift to 32 °C. Figure 3 shows that, although the effect was variable from one experiment to another, both eIF4GI and eIF4B underwent partial cleavage within 6–8 h following the activation of p53 in the Val135 cells, giving rise to discrete fragments. These changes were not seen in the Pro190 cells after the shift to 32 °C. The fragments that were generated correspond in size to the eIF4GI cleavage products N-FAG + M-FAG and M-FAG alone [27] (Fig. 3A,B) and to the eIF4B cleavage product DeIF4B [28,29] (Fig. 3C). These products have previously been observed in both human and mouse cells induced to undergo apoptosis in response to treatment with cyclo- heximide, anti-Fas (CD95) antibody, TNF-a,TRAIL, staurosporine or etoposide [24–29,39]. Partial cleavage was also seen in the case of the eIF4E binding protein 4E-BP1 in the Val135 cells (data not shown). Fig. 2. Activation of p53 results in rapid impairment of protein synthesis that precedes the loss of cell viability and irreversible commitment to cell death. (A) Exponentially growing Val135 and Pro190 cells were counted, transferred from 38 °Cto32°C and incubated for the times shown. Rates of protein synthesis per 10 5 cells were then measured by pulse-labeling the cells with [ 35 S]methionine (10 lCiÆmL )1 )for15min (two incubations, each in triplicate), as described in Materials and methods. Methionine incorporation in the Val135 cells is shown as a percentage of that in the Pro190 cells at the same temperature (s). The cell viabilities were determined by trypan blue exclusion and are plotted on the same time scale (d,m). (B) Val135 and Pro190 cells were extensively diluted and placed in multiwell plates such that an average of only one cell was present in every three wells of the plates. After incubation for various times at 32 °C, the cells were shifted back to 38 °C and allowed to proliferate. The wells were observed micro- scopically 10 days later and scored for the numbers of colonies formed. The data are the means ± the ranges of duplicate determinations. Dark-shaded bars, Pro190 cells; light-shaded bars, Val135 cells. Fig. 3. Activation of p53 causes cleavage of initiation factors eIF4GI and eIF4B. (A) Characterization of cleavage products of eIF4GI: Pro190 and Val135 cells were incubated at 32 °C for 15 h and extracts prepared and analyzed by immunoblotting for initiation factor eIF4- GI. The positions of migration of the intact factor ( 200 kDa), an intermediate cleavage product comprising the N-terminal and middle fragments of eIF4GI (N-FAG plus M-FAG) ( 150 kDa) and the middle fragment of eIF4GI (M-FAG) ( 76 kDa) [27] are indicated. (B) Time-course of cleavage of eIF4GI following activation of p53 at 32 °C. Pro190 and Val135 cells were incubated at 32 °Cforthetimes indicated and extracts were analyzed for the disappearance of intact eIF4GI and the appearance of M-FAG as in (A). (C) Time-course of cleavage of eIF4B following activation of p53 at 32 °C. Pro190 and Val135 cells were incubated at 32 °C for the times indicated and extracts were analyzed for the presence of eIF4B (80 kDa) and its cleavage product DeIF4B (60 kDa) as described previously [28,29]. No cleavage of eIF4B was observed when either cell line was maintained at 38 °C(seeFig.5C). Ó FEBS 2003 Regulation of protein synthesis by p53 (Eur. J. Biochem. 270) 3125 Effects of p53 on protein synthesis and initiation factor cleavages are independent of caspases To determine whether caspases are activated under the same conditions that result in the initiation factor cleavages we have assayed caspases-3, -8 and -9 in extracts from the two cell lines. As shown in Table 1, within 6 h at 32 °Cthe activities of all three caspases increased significantly in the Val135 cells but not in the Pro190 cells, reaching a maximum at about 20 h. In addition, we determined the extent of cleavage of two well characterized caspase substrates, PARP and the cyclin-dependent protein kinase inhibitor p27 KIP1 (Fig. 4A,B). Consistent with the activa- tion of caspase-3, processing of PARP to give rise to the characteristic p89 cleavage product was observed (Fig. 4A). A proportion of p27 KIP1 was also cleaved to produce a discrete fragment, Dp27 (Fig. 4B). Both the p53-induced increase in caspase-3 activity and the cleavages of PARP and p27 KIP1 were inhibited by the broad specificity caspase inhibitor z-VAD.FMK (Table 2, Fig. 4A,B). The above results show that, although cell viability does not decline until about 20 h (Fig. 2), p53 activation does cause increased caspase activity within 6 h. We therefore investigated whether caspase activity is responsible for the early changes in protein synthesis and the initiation factor cleavages that occur following the activation of p53. Figure 5(A,B) shows that the p53-induced inhibition of protein synthesis was completely resistant to treatment of the cells with z-VAD.FMK, both at early and late times after the temperature shift. Moreover, neither the cleavage of eIF4GI nor that of eIF4B was prevented by the caspase inhibitor, even when these cleavages involved only a relatively small fraction of the respective proteins (Fig. 5C). This was the case even though the z-VAD.FMK was able to block completely the activity of caspase-3 (Table 2), as well as that of caspases-8 and -9 (data not shown), and prevented the cleavages of PARP and p27 KIP1 induced by p53 activation (Fig. 4A,B). Moreover, the same z-VAD.FMK preparation inhibited the extensive cleavages of eIF4GI and eIF4B that occur in another apoptotic system, viz. Jurkat cells treated with an agonistic anti-Fas antibody [32,33] (Fig. 5C). In some experiments where more extensive cleavage of eIF4GI occurred z-VAD-FMK had a very slight protective effect but a substantial level of M-FAG was still generated in the presence of the caspase inhibitor Table 1. Activation of p53 rapidly enhances caspase activity. Pro190 and Val135 cells were incubated at 32 °C for the times indicated. Cell extracts were prepared and the activities of caspases-3, -8 and -9 were assayed as described in Materials and methods. The data are expressed as RFU per lg of protein and show the means ± the ranges of duplicate determinations. Cell line Caspase activity (RFUÆlg )1 protein) Caspase-8 Caspase-9 Caspase-3 Pro190 Val135 Pro190 Val135 Pro190 Val135 Hours at 32 °C 0 73.6 ± 6.7 63.9 ± 5.7 42.8 ± 5.5 53.6 ± 5.2 82.0 ± 3.6 127.7 ± 2.0 2 80.6 ± 1.9 89.5 ± 8.3 39.6 ± 4.7 62.5 ± 10.1 67.4 ± 0.1 168.3 ± 17.5 6 79.4 ± 2.9 117.6 ± 13.6 43.6 ± 2.0 94.3 ± 1.2 67.5 ± 0.1 413.8 ± 17.5 20 62.8 ± 2.1 230.3 ± 69.1 34.0 ± 0.7 213.0 ± 5.0 56.1 ± 3.1 966.6 ± 78.9 30 74.8 ± 8.4 243.9 ± 22.6 43.0 ± 8.5 163.3 ± 3.0 55.1 ± 1.4 577.5 ± 13.5 Fig. 4. Activation of p53 in MEL cells causes caspase-dependent cleavages of PARP and p27 KIP1 . (A) Pro190 and Val135 cells were incubated at 32 °C in the presence and absence of z-VAD.FMK for 15 h. Extracts were prepared and immunoblotted for (A) the apoptotic cleavage product of PARP (89 kDa) and (B) p27 KIP1 and its caspase cleavage product Dp27 as described previously [33–35]. Table 2. p53-induced caspase activity is sensitive to inhibition by z.VAD-FMK. Val135 cells were incubated at 38 °Corat32°Cfor6h in the presence and absence of the caspase inhibitor z.VAD-FMK (10 l M and 50 l M ). Extracts were prepared and assayed for caspase-3 activity by cleavage of the substrate Ac-DEVD.AMC as described in Materials and methods. The data are expressed as RFU per micro- gram of protein and are the means ± the ranges of duplicate deter- minations. Condition Caspase-3 activity (RFUÆlg protein )1 ) 38 °C 68.6 ± 4.8 32 °C 290.0 ± 5.1 32 °C plus z.VAD-FMK (10 l M ) 50.7 ± 1.6 38 °C plus z.VAD-FMK (50 l M ) 17.5 ± 1.6 3126 C. Constantinou et al. (Eur. J. Biochem. 270) Ó FEBS 2003 (Fig. 5D). These data therefore suggest that p53 regulates protein synthesis by mechanism(s) that do not require caspase activity and are consistent with the conclusion that the translational inhibition occurs independently of the induction of apoptosis. It is likely that other proteases are involved in causing the initiation factor cleavages and these may be responsible for regulating translation following p53 activation [40,41]. Incubation of the Val135 cells with a range of protease inhibitors, viz. the chymotrypsin inhibitor TPCK, the calpain inhibitors N-acetyl-Leu-Leu-Nle-CHO (ALLN), calpain inhibitor IV (z-LLY.FMK) and calpeptin (z-Leu- Nle-CHO), and the cathepsin B inhibitor z-FA.FMK, prevented neither the p53-induced cleavage of eIF4GI nor the inhibition of protein synthesis at 32 °C (data not shown). Further investigations utilizing a wider range of protease inhibitors will therefore be necessary to identify the enzyme(s) involved. Although the inhibition of protein synthesis by p53 activation can be dissociated temporally from the progress of apoptosis, we wanted to determine whether the preven- tion of cell death had an effect on the down-regulation of translation. To address this question we took advantage of the observation that treatment of cells either with z-VAD.FMK or with cytokines that function as survival factors inhibits p53-induced apoptosis [13,30,42–46]. In our hands, although z-VAD.FMK and the erythroid cell- specific survival factor erythropoietin (Epo) each had a marked antiapoptotic effect, both were required together to prevent completely the loss of viability of Val135 cells at 32 °C (Fig. 6A). In spite of this dramatic protective effect, however, neither z-VAD.FMK nor Epo, alone or in combination, showed any ability to rescue protein synthesis from p53-induced inhibition (Fig. 6B). This again suggests that the down-regulation of translation by p53 does not require the activity of caspases or other apoptotic mediators. It also indicates that the inhibition does not involve other pathways that are inactivated in the presence of Epo. Discussion The tumour suppressor protein p53 is activated in cells by a number of stresses, including UV irradiation, chemically induced DNA damage and hypoxia [47–50]. Activation of p53 results in a variety of cellular responses, notably inhibition of cell cycle progression and stimulation of DNA repair [51]. If p53 activity is sustained it can also lead to cell death by apoptosis [52]. Many of these effects require nuclear translocation of p53 and subsequent transcriptional activation of a large number of target genes [10,53]. However there is also evidence for direct cytoplasmic effects of activated p53, including association of the protein with Fig. 5. p53-induced translational inhibition and initiation factor cleavages do not require caspase activity. (A and B) Protein synthesis measurements. Pro190 and Val135 cells were incubated at 32 °Cfor(A)4hor6hor(B)15hinthepresenceandabsenceofz-VAD.FMK(50l M ) and protein synthesis was determined as described in Materials and methods. (C) Pro190 and Val135 cells were incubated at 32 °Cfor15hinthepresenceand absence of z-VAD.FMK and cell extracts were immunoblotted for eIF4GI or eIF4B and their cleavage products. As a positive control for the efficacy of the z-VAD.FMK, Jurkat cells were incubated for 2 h with or without an agonistic anti-Fas antibody [32], in the presence or absence of the same preparation of the inhibitor, and extracts were blotted for the same initiation factors. (D) Val135 cells were incubated at 38 °Cor32 °Cfor 6 h in the presence or absence of z-VAD.FMK as indicated. Cell extracts were immunoblotted for eIF4GI and its cleavage products. Ó FEBS 2003 Regulation of protein synthesis by p53 (Eur. J. Biochem. 270) 3127 mitochondria and ribosomes [15,16,54], and several studies have shown that pro-apoptotic effects of p53 do not necessarily require transcriptional transactivation activity of the protein [55–57]. Well documented reports have revealed a role for p53 in the control of translation of individual mRNA species such as those encoding cdk4, FGF-2 and even p53 itself [14,17– 21]. However the mechanisms responsible have not been elucidated. Our data show that p53 can also control the rate of global protein synthesis. Although the inhibition of translation precedes the impairment of cell cycle progression such an effect may ultimately contribute to the growth inhibitory effects of the activated tumour suppressor protein. Detailed analysis of the kinetics of the inhibition of translation has shown that this effect begins within 2–4 h of activating wild-type p53 [22] (manuscript in preparation). Thus it is unlikely that the regulation of protein synthesis is simply a consequence of either the impairment of cell cycle progression or the induction of apoptosis, both of which are associated with inhibition of translation in other systems [58–60]. However the question of whether common signal- ing pathways are involved in the control of translation and in the effects of p53 on the cell cycle and/or apoptosis will require the use of further mutants of p53 defective in inducing one or other of the latter effects. It is possible that the effects of p53 on protein synthesis are a result of new gene transcription events, although the early response time would tend to mitigate against this. Unfortunately we have been unable to use transcription inhibitors to investigate this possibility directly because such agents alone affect p53 function [61]. We have reported elsewhere that the extent of phos- phorylation of the inhibitor of polypeptide chain initiation factor eIF4E, 4E-BP1, is reduced following activation of p53 in the Val135 cells and that this results in sequestra- tion of eIF4E away from the eIF4F initiation complex [22]. No changes in the phosphorylation state of other key protein synthesis initiation factors such as eIF2a or eIF4E could be observed. The possibility that the dephosphory- lation of 4E-BP1 results in the inhibition of translation of specific mRNAs, including those known to be regulated at the translational level by p53, remains to be tested. The changes in 4E-BP1 function, in combination with the partial cleavages of eIF4GI and eIF4B reported here, may be sufficient to bring about the overall inhibition of protein synthesis by p53. However the present data do not address this issue directly. In many experiments a significant proportion of both eIF4GI and eIF4B remained intact following p53 activation; nevertheless it is possible that the cleavage products that accumulate could exert an inhibitory (dominant negative) effect on the activity of the remaining full-length protein. A further consequence of the cleavage of eIF4GI could be the stimulation of cap-independent translation. Fragments that are generated in apoptotic cells from both eIF4GI itself [62] and the related protein DAP5 [63,64] have been shown to enhance the utilization for translation of mRNAs with internal ribosome entry sites. At first sight the specific cleavages of initiation factors eIF4GI and eIF4B, both of which are known substrates for proteolysis in apoptosing cells, would seem to be in accord with the established pro-apoptotic effects of p53. These factors have been shown previously to be cleaved in cells undergoing apoptosis in response to treatment with anti-Fas antibody [28,32,33], cycloheximide [24,28], staurosporine or tumour necrosis factor a [26]. However, as shown in Figs 2 and 3, the initial down-regulation of translation, as well as the cleavages of eIF4GI and eIF4B, occurs at a time when there is little loss of cell viability and during a period when the p53-induced inhibition of cell growth is reversible [65]. This indicates that neither the translational inhibition nor the initiation factor modifications are simply consequences of apoptosis. Moreover these events are clearly not sufficient to commit the cells to death, although of course later changes that affect translation may be. Consistent with these conclusions, progression into apoptosis is not required for translational inhibition by p53 as essentially complete protection of the Val135 cells against death at 32 °Cby the combination of z.VAD-FMK and Epo did not rescue protein synthesis (Fig. 6). Both eIF4GI and eIF4B can be cleaved by caspase-3 in vivo and in vitro [27,29]. As caspase-3 is activated in the Val135 cells following the temperature shift, and the cleavage products of the two initiation factors appear to be Fig. 6. The p53-induced inhibition of protein synthesis can be dissociated from apoptosis. (A) Pro190 and Val135 cells were incubated at 38 °Cor 32 °C for 48 h in the absence or presence of Epo (10 unitsÆmL )1 )and/ or z.VAD-FMK (10 l M )asindicated.Attheendofthisperiodcell viability was determined by trypan blue exclusion. (B) Cells were incubated as in (A). After 24 h, protein synthesis was monitored by the incorporation of [ 35 S]methionine (10 lCiÆmL )1 ) into acid-insoluble material during the last 1 h of incubation. The data are expressed as a percentage of the incorporation in Pro190 cells incubated at 38 °Cin the absence of Epo and z.VAD-FMK. 3128 C. Constantinou et al. (Eur. J. Biochem. 270) Ó FEBS 2003 very similar, if not identical, to those seen as a result of caspase-dependent degradation in other systems, we were surprised to find that the appearance of M-FAG and DeIF4B was not inhibited by the broad specificity caspase inhibitor z.VAD-FMK. This was not due to a failure of the latter to act on MEL cells as the compound inhibited the activation of caspase-3 and completely blocked the cleavage of the caspase substrates PARP and p27 KIP1 in Val135 cells shiftedto32°C.Moreoverthesamez.VAD-FMKprepar- ation was effective in inhibiting the cleavage of eIF4GI and eIF4B in Jurkat cells treated with anti-Fas. Along with an inability to prevent the factor cleavages in the Val135 cells z.VAD-FMK was also unable to prevent the overall inhibition of protein synthesis. Although we cannot rule out the possibility that eIF4GI and eIF4B are cleaved by caspase(s) that are at least partially active even in the presence of z.VAD-FMK [66] it is possible that other proteases that are activated directly or indirectly by p53 are responsible [40,41]. Such alternative pathways may also operate in other systems. Whereas p53 is required for radiation-induced neuronal cell death, caspase activity is not required for this process [67]. Several studies have established the phenomenon of caspase-independent cell death. Moreover noncaspase proteases are involved in some forms of apoptosis mediated by p53 and other pathways, and specific protein cleavages occur in some cases [68–72]. Morley and Pain [73] reported that eIF4GI and eIF4GII can be cleaved by a z.VAD-FMK-resistant mech- anism in cells undergoing apoptosis in response to treatment with the immunosuppressant drugs FTY720 and cyclo- sporin A. If noncaspase mediated proteolytic events are responsible for the cleavage of eIF4GI and eIF4B the enzyme(s) involved must presumably act on sites that are identical or very close to those targeted by the caspases [27,28]. These sites may lie in relatively accessible or unstructured regions of the proteins. In spite of using a wide range of protease inhibitors we have not yet identified the protease(s) responsible for the initiation factor cleavages following p53 activation. The effects of p53 on the translational machinery are very similar to those seen following treatment of cells with DNA damaging agents such as etoposide, mitomycin-C or cisplatin [32,39,74]. Common features include the caspase- independent nature of the inhibition of overall translation, the lack of effect on eIF2a phosphorylation and, in contrast, the marked dephosphorylation of 4E-BP1 [22]. These observations suggest that the effects of DNA-damaging agents on translation could be mediated, at least in part, by p53. The p53-regulated effects we observe are also similar to those seen following inhibition of proteasome activity [75]. Proteasome inhibition not only induces p53-dependent apoptosis [76–78] but also causes dephosphorylation of 4E-BP1 and the cleavage of initiation factors, effects which are partially caspase-independent (S. Morley, personal communication). Cyclosporin A, which can induce eIF4G cleavage [73], also inhibits proteasome activity [79,80]. Moreover, inhibition of proteasome-mediated proteolysis induces p53 expression and caspase-independent apoptosis [78,81]. Another potential mechanism of action of p53 may involve signaling by ceramide as a second messenger. Ceramide causes caspase-independent apoptosis and also induces p53 in at least one system [82,83]. Irradiation- induced DNA damage activates ceramide production [84], and p53 is required for the induction of ceramide by some cell stresses [85]. Whether the tumour suppressor protein is required for ceramide-induced growth inhibition and apoptosis remains controversial however, [85–87]. If p53 functions upstream of ceramide then the latter may indeed contribute to the down-regulation of translation observed in this study. However ceramide has been reported to activate the eIF2a-specific protein kinase PKR and thereby inhibit translation [88], whereas p53 activation has no effect on eIF2a phosphorylation [22]. Few other studies of the effects of ceramide on protein synthesis or initiation factor modifications have been reported and the possibility of regulation by this second messenger in cells expressing active p53 remains to be evaluated. In summary, we have reported the novel observation that activation of p53 results in the caspase-independent down- regulation of translation, together with the cleavages of at least two polypeptide chain initiation factors that are critical for protein synthesis. Moreover, we have shown that these events are not simply the consequences of p53-induced apoptosis and indeed occur independently of this process. Further details of the mechanisms involved await future study. Acknowledgements This research was supported by grants to M. J. Clemens from the Wellcome Trust (056778), the Leukaemia Research Fund and Glaxo- Wellcome and by grants to J. Hensold from the Office of Research and Development, Medical Research Service, Department of Veterans’ Affairs and the NIH (DK43414). J. Hensold was also funded during a period of sabbatical leave by an award from Burroughs-Wellcome. C. Constantinou is supported by a PhD studentship from the Cancer Prevention Research Trust, with additional funding from the AG Leventis Foundation and an Overseas Research Scholarship from Universities UK. M. Bushell is supported by a Fellowship from The Wellcome Trust (063233). References 1. Johnson, P., Gray, D., Mowat, M. & Benchimol, S. (1991) Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells. Mol. Cell Biol. 11, 1–11. 2. Burns, T.F. & El-Deiry, W.S. (1999) The p53 pathway and apoptosis. J. Cell. Physiol. 181, 231–239. 3. Sionov, R.V. & Haupt, Y. (1999) The cellular response to p53: the decision between life and death. Oncogene 18, 6145–6157. 4. Appella, E. 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