Translational Control of Programmed Cell Death: Eukaryotic ...

MOLECULAR AND CELLULAR BIOLOGY, Nov. 1996, p. 6573–6581 0270-7306/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 16, No. 11

Translational Control of Programmed Cell Death: Eukaryotic Translation Initiation Factor 4E Blocks Apoptosis in Growth-Factor-Restricted Fibroblasts with Physiologically Expressed or Deregulated Myc VITALY A. POLUNOVSKY,1 IGOR B. ROSENWALD,2 ANNIE T. TAN,1 JAMES WHITE,3 LAN CHIANG,1 NAHUM SONENBERG,4 AND PETER B. BITTERMAN1* Pulmonary and Critical Care Division, Department of Medicine,1 and Department of Laboratory Medicine and Pathology,3 University of Minnesota Medical School, Minneapolis, Minnesota 55455; Division of Health Sciences and Technology, Harvard-Massachusetts Institute of Technology, Cambridge, Massachusetts 021392; and Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y64 Received 14 February 1996/Returned for modification 5 April 1996/Accepted 22 July 1996

There is increasing evidence that cell cycle transit is potentially lethal, with survival depending on the activation of metabolic pathways which block apoptosis. However, the identities of those pathways coupling cell cycle transit to survival remain undefined. Here we show that the eukaryotic translation initiation factor 4E (eIF4E) can mediate both proliferative and survival signaling. Overexpression of eIF4E completely substituted for serum or individual growth factors in preserving the viability of established NIH 3T3 fibroblasts. An eIF4E mutant (Ser-53 changed to Ala) defective in mediating its growth-factor-regulated functions was also defective in its survival signaling. Survival signaling by enforced expression of eIF4E did not result from autocrine release of survival factors, nor did it lead to increased expression of the apoptosis antagonists Bcl-2 and Bcl-XL. In addition, the execution apparatus of the apoptotic response in eIF4E-overexpressing cells was found to be intact. Increased expression of eIF4E was sufficient to inhibit apoptosis in serum-restricted primary fibroblasts with enforced expression of Myc. In contrast, activation of Ha-Ras, which is required for eIF4E proliferative signaling, did not suppress Myc-induced apoptosis. These data suggest that the eIF4E-activated pathways leading to survival and cell cycle progression are distinct. This dual signaling of proliferation and survival might be the basis for the potency of eIF4E as an inducer of neoplastic transformation. macromolecular synthesis. An increased rate of mRNA translation is an essential component of the proliferative response (5, 34). The rate-limiting step of translation initiation is transfer of the mRNA to the 40S ribosomal subunit. This event is mediated by the cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E) (16, 32, 60). Growth factors stimulate eIF4E production through a Myc-mediated transcriptional mechanism (52). Growth-factor-induced phosphorylation events are also essential for the translation-promoting function of eIF4E. These include growth-factor-dependent phosphorylation of eIF4E (10, 23, 33, 47) and phosphorylation of its inhibitory binding protein 4E-BP1 (PHAS-I) (13, 15, 30, 37, 39). When phosphorylated, 4E-BP1 releases eIF4E from a functionally inactive complex, enabling it to activate translation (30, 39). Although required for translation of all capped mRNAs, increased expression of eIF4E leads to a selective increase in translation of certain mRNA species (reviewed in references 32 and 46). Many of these mRNAs direct production of cell cycle-related proteins that are typically downregulated in quiescent cells. Enforced overexpression of eIF4E results in selective enhancement of key regulators of cell cycle progression, including ornithine decarboxylase (55) and cyclin D1 (50, 51). As a result, overexpression of eIF4E decreases the growth factor requirement for cell cycle progression and mimics the ability of growth factors to stimulate cell proliferation (58), as well as morphogenesis (25). In addition, enhanced expression of eIF4E in established or primary fibroblasts causes malignant transformation (27, 29). A single amino acid substitution at

Normal diploid fibroblasts deprived of serum or polypeptide growth factors exit the cell cycle and persist in a quiescent state. When quiescent cells are stimulated to enter the cell cycle by viral oncogenes (31) or by enforced expression of cellular growth-promoting proteins such as c-Myc (2, 8; reviewed in reference 38), E2F (1, 17, 68), or cyclin A (18, 35), they do not progress toward mitosis but undergo programmed cell death. Similarly, the relaxation of negative growth control that occurs during preneoplastic progression of fibroblasts toward immortalization (42, 62) or by decreased retinoblastoma protein expression (1, 57) leads to apoptosis in response to growth factor withdrawal. Along these lines, peptide growth factors such as platelet-derived growth factor (PDGF) and epidermal growth factor can signal both cell cycle entry and apoptosis in fibroblasts deprived of progression-type growth factors (24). These findings support the idea that cell cycle transit is a potentially lethal condition unless specific rescue factors prevent this fate (20, 43, 44). Therefore, a mitogenic stimulus must coordinately activate metabolic pathways directing both proliferation and survival in order to result in cell division without death. While recent advances in our understanding of the cell division cycle reveal how cells integrate proliferative signals with the cell cycle machinery, the molecular mechanisms and regulatory pathways involved in growth factor survival signaling remain uncertain. The quiescent state is characterized by a reduced rate of * Corresponding author. 6573

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position 53 in the eIF4E protein (Ser-53 to Ala-53) impairs both its mitogenic and transforming activities (reviewed in references 46 and 60). Here we show that the overexpression of eIF4E can substitute for serum or individual growth factors in blocking the apoptotic program activated in growth-factor-restricted immortal fibroblasts or in primary fibroblasts expressing deregulated Myc. This function of eIF4E requires the presence of its Ser-53 and does not correlate with the levels of survival proteins Bcl-2 and Bcl-XL. Our data also indicate that the survival function of eIF4E is not associated with its growth-promoting and transforming activities. Thus, eIF4E-mediated translational control may participate in two independent regulatory pathways involved in growth factor signaling, i.e., stimulation of cell cycle progression and activation of a salvage pathway that suppresses apoptosis in cycling cells. MATERIALS AND METHODS Cell lines and cell culture. Mouse neonatal lung fibroblasts (MLF) were prepared by dissecting and mincing lungs of newborn BALB/c mice. The explants were cultivated in Dulbecco’s modified Eagle medium (DMEM) supplemented with 20% fetal calf serum (FCS) for 5 days in tissue culture dishes (Falcon). Explants were removed, and the cells which had migrated from the explants were enzymatically detached (0.25% trypsin), with cultures continued in the same medium. Parental NIH 3T3 fibroblasts and cells constitutively expressing wildtype eIF4E (eIF4E wt cells) or a functionally impaired mutant whose serine 53 residue was replaced by alanine (eIF4E Ala cells) were described previously (4, 28). NIH 3T3 cells carrying the same retroviral construct lacking eIF4E sequences served as a control (NIH 3T3 neo cells). Parental rat embryo fibroblasts (REF) and their derivatives constitutively expressing c-myc, v-myc, Ha-ras, or eIF4E or coexpressing combinations of these genes were generated as described previously (29). All cells were maintained in DMEM supplemented with 10% FCS, 100 U of penicillin per ml, 100 U of streptomycin per ml, and 250 ng of amphotericin B per ml (Gibco). In addition, those cell lines carrying retroviral vectors or oncogeneexpressing plasmids were cultivated in DMEM with all of these supplements plus G418 (400 mg/ml). For studies of cell survival under growth-factor-restricted conditions, exponentially growing cultures were shifted to DMEM containing either 0.1 or 0.5% FCS for various times or were cultivated in a serum-free chemically defined medium (DMEM-F12 mixture supplemented with bovine serum albumin [BSA] [0.1 mg/ml], Fe-transferrin [10 mg/ml], selenium [1028 M], and linolic acid [3 3 1026 M]). Cell population dynamics were assessed by counting cells with an electronic particle counter. The cell cycle distribution was monitored by flow cytometric analysis of DNA content (41). Entry into S phase was identified by analysis of bromodeoxyuridine (BrdU) incorporation into DNA. Cells were exposed to 100 mM BrdU (Sigma) for 2 h, fixed in 70% ethanol, and incubated for 30 min with anti-BrdU fluorescein isothiocyanate-conjugated antibody (Boehringer Mannheim, Indianapolis, Ind.) diluted 1:50, and the signal was quantified by flow cytometry. The staining with BrdU-specific antibodies was done according to the protocol provided by the antibody manufacturer. Fixed cells were resuspended in 1 ml of chilled 0.1 M HCl containing 0.5% Triton X-100 for 10 min on ice to extract histones, washed in distilled water, and incubated at 1008C for 10 min to denature DNA. Before being stained, the cells were washed in phosphatebuffered saline (PBS) containing 0.5% Triton X-100 and resuspended in 100 ml of PBS containing 0.1% BSA and either anti-BrdU fluorescein isothiocyanateconjugated antibody or the same amount of control isotype immunoglobulin (PharMingen). After incubation for 30 to 45 min at room temperature, the cells were subjected to quantitative flow cytometry. Cell death analysis. Cell viability was estimated by staining with trypan blue. To define the mode of cell death, morphology was examined by phase-contrast and transmission electron microscopy as described previously (40, 41). In addition, apoptosis-associated DNA fragmentation was determined by agarose gel electrophoresis (40) and by in situ staining of apoptotic nuclei by the terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method (12) and was quantified by flow cytometric analysis of the percentages of cells with hypodiploid DNA content (40). For specific staining of apoptotic nuclei by the TUNEL method, cell monolayers were rinsed with PBS, and cells were fixed (10% buffered formalin, 48C, 15 min). The staining procedure was performed as described previously (12). A terminal transferase (Boehringer Mannheim) reaction was performed with fixed cells to incorporate biotinylated dUTP (Enzo Diagnostics). Color development was accomplished with ExtrAvidin-Peroxidase (Sigma) stained with 3-amino-9-ethylcarbazole (AEC) (Sigma). For flow cytometric determination of DNA degradation, 2 3 105 cells were seeded onto 100-mm-diameter dishes 20 h prior to alteration of the serum concentration. At each time point analyzed, floating cells were collected, com-

MOL. CELL. BIOL. bined with adherent cells (released with trypsin), and fixed with ethanol (70%, 48C) for 1 h. The fixed cells were washed with PBS and incubated in propidium iodide stain mixture (50 mg of propidium iodide per ml, 0.05% Triton X-100, 0.1 mM EDTA, and 100 U of RNase per ml in PBS). After incubation (45 min, 378C), the DNA content was determined by quantitative flow cytometry (BectonDickinson FACS Star Plus). Results were tabulated as the means 6 standard deviations from three to five separate experiments, and the histograms shown are from a single representative experiment. In each experiment, all conditions were examined in duplicate or triplicate. Immunoprecipitation and Western blot (immunoblot) analysis. Bcl-2 was evaluated by immunoprecipitation of 700 mg of crude cell lysate with hamster anti-mouse Bcl-2 (1:300) (clone 3F11; PharMingen). After incubation (48C, 16 h), immune complexes were bound to protein G-Sepharose, washed, solubilized, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (14%) under reducing conditions, and blotted onto a nitrocellulose membrane. Immunodetection was performed with 3F11 (1:100) followed by horseradish peroxidase-labeled goat anti-hamster immunoglobulin G with diamindobenzidine used as the color substrate. To examine Bcl-XL, 140 mg of crude cell lysate was subjected to SDS-PAGE (12%) under reducing conditions and blotted onto a nitrocellulose membrane. Immunodetection was accomplished with an affinity-purified rabbit anti-murine Bcl-XL antibody (2.5 mg/ml) (kindly provided by T. W. Behrens, Department of Medicine, University of Minnesota) followed by horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G with diaminobenzidine used as the color substrate. In vitro translation samples generated with and without murine Bcl-XL mRNA were included as controls. Western blot analysis of eIF4E expression was performed as described previously (50).

RESULTS Overexpression of eIF4E prevented death of serum-restricted NIH 3T3 fibroblasts. We examined cell population kinetics under growth-restricted conditions among cells with different growth patterns. These included transformed NIH 3T3 fibroblasts carrying a retroviral pMV7-4E construct containing wild-type eIF4E sequences (eIF4E wt cells), NIH 3T3 cells carrying the same construct without eIF4E (NIH 3T3 neo cells), and primary cultures of MLF as nonimmortalized controls. In addition, we employed NIH 3T3 cells infected with the same expression vector containing a mutant eIF4E DNA fragment (eIF4E Ala cells). These cells express approximately the same level of eIF4E as eIF4E wt cells (27), but a functionally important residue, Ser-53, was replaced with Ala. Growth restriction was achieved by culturing cells in medium lacking isoleucine or in medium containing a low concentration of serum. Each of the cell lines tested revealed similar cell population growth rates when deprived of isoleucine in serumreplete medium (10% FCS), with cell numbers increasing briefly and then reaching a plateau (Fig. 1A). Under serumrestricted culture conditions, the numbers of eIF4E wt cells and MLF increased modestly, while the numbers of NIH 3T3 neo and eIF4E Ala cells declined (Fig. 1B). NIH 3T3 neo cells, which expressed a physiological level of eIF4E, progressively detached from the substratum and lost viability (.90% trypan blue positive [not shown]), consistent with previous reports indicating that established murine fibroblasts lines die when deprived of serum (53, 63–65). eIF4E Ala cells also died when deprived of serum. In contrast, serum-restricted MLF and eIF4E wt cells remained viable (,2% trypan blue positive). Our findings with isoleucine-deficient medium indicated that serum-restricted NIH 3T3 fibroblasts did not die from cell cycle arrest per se but died because of the absence of specific serum survival factors. Overexpression of eIF4E rescued serum-restricted cells. Serum-induced fluctuations in the level of eIF4E were associated with vulnerability to serum restriction. The basal level of eIF4E expression in MLF was low and did not depend significantly on the serum concentration (Fig. 2). Following serum withdrawal, NIH 3T3 neo cells downregulated eIF4E to the basal level. These oscillations in eIF4E expression did not depend on the presence of isoleucine in the medium (data not shown), suggesting that the observed levels of eIF4E were

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TRANSLATIONAL CONTROL OF PROGRAMMED CELL DEATH

FIG. 1. Cell numbers under growth-restricted conditions. Primary MLF and NIH 3T3 cells carrying either an eIF4E PMV7 retroviral construct containing wild-type eIF4E (eIF4E wt), a mutant gene (eIF4E Ala), or an empty vector (NIH 3T3 neo) were seeded into six-well dishes (105 cells per well) in DMEM plus 10% FCS. After 3 h, the cells were washed three times with DMEM lacking serum, and cultures were continued in DMEM lacking isoleucine (2IL) and supplemented with 10% dialyzed FCS (A) or in isoleucine-replete DMEM (1IL) supplemented with 0.1% FCS (B). At the indicated times, cells were released from the substratum with trypsin and enumerated with an electronic particle counter. Shown are relative cell numbers as a function of time.

associated with changes in serum concentration rather than with cell cycle progression. Thus, unlike primary fibroblasts, established NIH 3T3 cells required either serum or upregulated eIF4E for their survival and increased their level of eIF4E in response to serum. NIH 3T3 cells underwent apoptosis with low serum concentrations and required functionally active eIF4E to survive. We sought to establish the manner of cell death and to examine whether the translational activity of eIF4E was required to rescue serum-restricted NIH 3T3 cells. We therefore examined morphology and DNA integrity in NIH 3T3 neo cells, eIF4E wt cells, or eIF4E Ala cells. Analysis of NIH 3T3 cells revealed that they die by apoptosis (69). Serum-restricted NIH 3T3 neo cells manifested a timedependent increase in peripheral chromatin condensation, nuclear fragmentation, and degradation of cytoplasmic organelles (Fig. 3A). By the TUNEL method, there was specific staining in cells with morphological features typical of apoptosis as well as in apoptotic bodies (Fig. 3B). Agarose gel electrophoresis (Fig. 3C) and quantitative flow cytometry (Fig. 3D) demonstrated progressive degradation of DNA in serum-restricted

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NIH 3T3 neo cells, whereas DNA from eIF4E wt cells remained intact. Cells overexpressing mutant eIF4E manifested levels of apoptosis intermediate between those of parental cells and of cells overexpressing wild-type eIF4E (Fig. 3C), ultimately dying in low-serum medium after 11 days (Fig. 3D). In contrast, eIF4E wt cells, under the same conditions, formed characteristic densely packed foci (Fig. 3E). This result demonstrated a functional concordance between translational activity and suppression of apoptosis. Thus, rescue of NIH 3T3 cells from apoptosis in low-serum medium depended upon the presence of functionally active eIF4E protein. Cells overexpressing eIF4E did not require growth factors for survival and did not secrete autocrine survival factors. The capacity of eIF4E to preserve cell viability under low-serum conditions could result from its ability to substitute for specific peptide growth factors required for survival or to substitute for other, unidentified trophic signals provided by serum. To address this issue, NIH 3T3 neo cells or eIF4E wt cells were incubated in defined medium supplemented with insulin-like growth factor-1 (IGF-1), PDGF, or both. IGF-1 (20 ng/ml) or PDGF (5 ng/ml) individually decreased the proportion of apoptotic cells by 70 to 85%, and both together eliminated the apoptotic response. On the basis of this result, the ability of eIF4E to block apoptosis could have been mediated through specific apoptotic regulatory pathways or by increasing autocrine production of survival factors (20, 22). When NIH 3T3 neo cells were transferred into serum-restricted medium conditioned for 72 h by eIF4E wt cells, there was no effect on the proportion of cells undergoing apoptosis (Fig. 4). Together these data indicated that apoptosis in response to serum restriction was due to growth factor withdrawal and did not result from serum nutrient deprivation. In addition, the ability of eIF4E to replace the requirement for exogenous growth factors in the maintenance of cell viability could not be accounted for by production of autocrine survival factors. Overexpression of eIF4E did not disable the execution apparatus of the apoptotic pathway. The observed interdiction of apoptosis by eIF4E could have targeted either the regulatory arm or the effector arm of the apoptotic pathway. The effector arm of the programmed death pathway includes factors leading to cell disintegration, as well as safeguard mechanisms preventing apoptosis until lethal signaling ensues. It requires the function of a protease represented by the product of the ced-3 gene in nematodes or the homologous interleukin-1b-converting enzyme gene family in mammals (reviewed in references 26 and 61). It is inhibited by the product of the ced-9 gene in nematodes, which is homologous to the Bcl-2 family of proteins in

FIG. 2. Expression of eIF4E in murine fibroblast lines. Cells were seeded in 100-mm-diameter dishes and cultured for 3 days in DMEM plus 10% FCS. The cells were incubated for an additional 48 h in fresh DMEM containing either 10 or 0.1% FCS before they were lysed, and expression of eIF4E was examined by Western blotting.

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mammals (reviewed in references 19 and 45). To evaluate the integrity of apoptotic effector pathways, we exposed NIH 3T3 neo cells and eIF4E wt cells to camptothecin (0.75 mM), a topoisomerase I inhibitor (59), or to a monoclonal antibody blocking the interaction of cell surface proteoglycan CD44 with the substratum (56) (Fig. 5A). In both cell populations, 57 to

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FIG. 3. Functionally active eIF4E inhibits apoptosis in serum-restricted NIH 3T3 fibroblasts. NIH 3T3 cells expressing wild-type eIF4E (eIF4E wt), a mutant eIF4E in which serine 53 was replaced by alanine (eIF43 Ala), or empty vector (NIH 3T3 neo) were analyzed. (A) Ultrastructural features of NIH 3T3 neo cells as a function of time in DMEM plus 0.1% FCS (panels: 1, 0 h; 2, 24 h; 3, 48 h; 4, 72 h). Cells were fixed and processed for electron microscopy as described previously (67). (B) TdT reaction in situ (TUNEL) in NIH 3T3 fibroblasts incubated in DMEM with 0.1% FCS for 48 h. Cells were fixed with 4% buffered formalin for 15 min and incubated in the presence of terminal transferase and biotinylated dUTP. TUNEL-positive cells (lower right) reveal condensed chromatin and cytoplasmic blebbing. Apoptotic bodies (left) contain TUNEL-positive nuclear fragments. (C) Agarose gel electrophoresis of DNA isolated from NIH 3T3 neo, eIF4E Ala, and eIF4E wt cells after culture for 72 h in DMEM plus 0.1% FCS. Genomic DNA was isolated from 5 3 106 cells and subjected to electrophoresis and ethidium bromide staining. (D) Flow cytometric analysis of DNA content as a function of time in DMEM plus 0.1% FCS. At the indicated times, suspended and attached cells were combined, washed in PBS, and fixed in 70% ethanol. Cells were stained with propidium iodide, and DNA was quantified. The percentage of cells with hypodiploid DNA (A) or replicating DNA (S) are presented for NIH 3T3 neo, eIF4E Ala, and eIF4E wt cells. Two independent experiments showed similar results. (E) Phase-contrast appearance of eIF4E Ala (panel 1) and eIF4E wt (panel 2) cells cultured in DMEM plus 0.5% FCS for 11 days. NIH 3T3 neo cells had all detached from the substratum and died within 5 days and are therefore not shown. Magnification, 3200.

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FIG. 4. Growth factors or eIF4E is required to preserve fibroblast viability in defined medium. NIH 3T3 fibroblasts were cultured in chemically defined medium alone or supplemented with PDGF BB (5 ng/ml), IGF-1 (20 ng/ml), or both. Conditioned medium (CM) to examine for the secretion of autocrine survival factors was produced by culturing eIF4E wt cells in defined medium lacking growth factors (GF) for 72 h. Shown are the percentages of apoptotic cells after 48 h of culture under the conditions indicated.

68% of the cells underwent apoptosis in response to camptothecin, and 78 to 84% of the cells underwent apoptosis after incubation with the anti-CD44 antibody. These data indicated that the apoptotic machinery in eIF4E wt cells was intact. Overexpression of eIF4E did not increase the levels of the survival proteins Bcl-2 and Bcl-XL. To directly determine if eIF4E selectively increased the translation of Bcl-2 family proteins, we measured the steady-state levels of two family members, Bcl-2 and Bcl-XL (3, 9, 54, 66). Immunoblot analysis of NIH 3T3 neo and eIF4E wt cells revealed similar amounts of Bcl-2 in the two cell lines, independent of serum concentration (Fig. 5B). Fluorescence-activated cell sorter (FACS) analysis of cycling NIH 3T3 neo and eIF4E wt cells revealed similar distributions of Bcl-2 expression and DNA content, with no discernible relationship between these two parameters (data not shown). Neither immunoblot nor FACS analysis of NIH 3T3 neo cells and eIF4E wt cells demonstrated any detectable Bcl-XL, which was easily detected in an in vitro translation system serving as a positive control (Fig. 5B). This indicated that eIF4E-mediated inhibition of apoptosis was not related to a direct increase in Bcl-2 or Bcl-XL expression. Overexpression of eIF4E abolished Myc-induced apoptosis. Since Myc has been implicated as an upstream participant both in transcriptional activation of eIF4E (52) and in the apoptotic response of growth-factor-restricted fibroblasts (8, 14), we examined the ability of eIF4E to suppress apoptosis in the context of physiologically regulated or upregulated Myc. To study this phenomenon against a nonimmortal background, we used diploid REF instead of NIH 3T3 cells. The cells were transfected with v-Myc or eIF4E or cotransfected with both. As expected for diploid fibroblasts, serum restriction for 24 h led to a modest induction of apoptosis (Table 1). Constitutive overexpression of v-Myc conferred on fibroblasts a fourfold

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increase in susceptibility to apoptosis in response to serum restriction. In contrast, similar to NIH 3T3 cells overexpressing wild-type eIF4E, diploid fibroblasts overexpressing eIF4E (REF eIF4E cells) were resistant to serum withdrawal. Overexpression of eIF4E virtually eliminated Myc-driven apoptosis. Overexpression of eIF4E stimulates DNA synthesis and induces Ras-dependent malignant transformation (27, 28). We therefore examined whether upregulation of Ras could reproduce the antiapoptotic effect of eIF4E or whether the survival pathway activated by eIF4E was distinct from those promoting DNA synthesis and transformation. REF transfected with c-Ha-ras (REF Ha-ras cells) manifested a transformed phenotype following selection in G418 and synthesized DNA in medium (DMEM plus 0.75% FCS) unable to sustain the proliferation of control fibroblasts (REF neo cells) (Table 1). When REF Ha-ras cells were further serum restricted (DMEM plus 0.5% FCS), they manifested an apoptotic response similar to that of REF neo cells and 10-fold greater than that of REF eIF4E wt cells. The same experiment carried out with highly transformed REF cotransfected with Ha-ras and v-myc led to a robust proliferative response; however, the apoptotic response to serum restriction was not significantly attenuated (25.5 versus 29.6%; P . 0.3). We compared the transfected fibroblast populations for their apoptotic responses and proliferative capacities and for the presence or absence of a transformed phenotype. We found no relationship between cell death and the ability of oncogenes to stimulate DNA synthesis in media with low serum concentrations. Similarly, apoptosis and expression of a malignant phenotype were not closely linked (Table 1). These data indicated that upregulation of endogenous Ras, or both Ras and Myc, although sufficient to promote cell proliferation and transformation, was not sufficient to inhibit apoptosis in growth-factor-restricted cells. Therefore, neither acquisition of a transformed phenotype by itself nor the ability to proliferate in low-serum medium was sufficient to confer resistance to the induction of apoptosis by growth factor restriction. DISCUSSION In prior studies we found that serum or PDGF induced increased expression of eIF4E in growth-factor-restricted NIH 3T3 fibroblasts (48–50). Overexpression of eIF4E mimics the physiological effect of growth factors in the activation of cell proliferation (58) and morphogenesis (25). Here we show that overexpression of eIF4E functionally substitutes for serum or individual growth factors in maintaining the viability of fibroblasts expressing either physiologically regulated or constitutively upregulated Myc. The single amino acid substitution Ser-53 to Ala blocks not only the mitogenic (58) and transforming (27) activities of eIF4E but also its survival activity (present study). These findings suggest that eIF4E might act as a growth factor sensor that integrates both proliferative and survival signaling with the cell cycle engine. This integration produces an output dependent on the translational activity of eIF4E that suppresses apoptosis. In our studies we found that the ability of a fibroblast cell line to change its expression of eIF4E in response to the addition or withdrawal of serum was related to its propensity to undergo apoptosis when growth factor restricted. Neither expression of eIF4E nor viability was significantly changed when primary fibroblasts were shifted from high to low serum concentrations and back again. In contrast, established NIH 3T3 cells express a greater level of eIF4E in high-serum medium than under serum-restricted conditions and undergo apoptotic death upon growth factor withdrawal. Enforced upregulation

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MOL. CELL. BIOL. FIG. 5. Overexpression of active eIF4E does not disrupt the execution apparatus or deregulate Bcl-2 and Bcl-XL. (A) Induction of apoptosis in eIF4E wt cells. NIH 3T3 neo and eIF4E wt cells were cultured for 24 h either with camptothecin (CAM) (0.75 mM; Sigma) or with anti-CD44 antibody (4 mg/ml; The Binding Site Limited) in DMEM plus 10% FCS. Cells incubated in DMEM plus 10% FCS served as negative controls. Shown are the proportions of cells with hypodiploid DNA as determined by FACS analysis. (B) Expression of Bcl-2 and Bcl-XL. Immunodetection was carried out in logarithmic and serum-restricted cultures as described in Materials and Methods. IVT, in vitro translation system; mbcl-xL, murine Bcl-XL.

of eIF4E permits survival of these cells when they are serum restricted. Thus, there is an apparent relationship between the cellular requirement for growth factors to survive and upregulation of eIF4E in response to growth factors. This relationship is in accord with the notion that eIF4E mediates physiological

TABLE 1. Apoptosis, DNA synthesis, and expression of a transformed phenotype in serum-restricted REF cell lines REF cell linea

REF REF REF REF REF REF a

neo v-myc elF4E Ha-ras v-myc/elF4E v-myc/Ha-ras

Apoptotic cells (%)b

BrdU uptake (% cells in S)c

10.2 29.6 0.9 8.9 1.9 25.5

1.8 27.8 23.2 35.7 58.6 53.4

Transformed phenotyped

2 2 1/2 1/2 1 1

Cell lines were produced as described previously (27). Cells were incubated in medium containing 0.5% FCS for 24 h, and apoptosis was quantified as described in Materials and Methods. c Uptake of BrdU was determined by FACS analysis of cells incubated in medium containing 0.75% FCS for 48 h and exposed to 100 mM BrdU for 2 h before fixation. d The transformed phenotype included typical cell morphology, the ability to form colonies in soft agar, growth pattern in vitro, and tumorigenicity in nude mice (27–29). 1/2, cells displayed malignant transformation following selection in medium containing G418 and a normal phenotype in the absence of selection. b

growth factor survival signaling by suppressing an underlying apoptotic program activated in immortalized fibroblasts by serum deprivation. One possible mechanism of eIF4E-activated survival signaling is that it might stimulate the secretion of growth factors that support survival in serum-free medium (20). This notion is consistent with our previous data that subconfluent cultures of eIF4E wt cells appear to be quiescent in serum-restricted medium but resume proliferation after 6 days (50), suggesting that eIF4E might stimulate the synthesis of autocrine growth factors that gradually accumulate in the culture medium. However, here we show that medium conditioned for up to 3 days by eIF4E wt fibroblasts did not promote survival of the parental NIH 3T3 cells. These findings are most consistent with the possibility that fibroblasts overexpressing eIF4E possess constitutively activated metabolic pathways that enable them to survive in the absence of extracellular survival signals. We interpret these findings to indicate that under physiological conditions, growth factors might promote cell survival by activating eIF4E-mediated salvage pathways keeping the intrinsic cell cycle-related death program suppressed. Previous studies indicate the importance of Ser-53 for the activity of eIF4E (reviewed in references 46 and 60). Mutation of Ser-53 to Ala impairs the ability of overexpressed eIF4E to activate cyclin D1 (50, 51) and to promote tumorogenesis in NIH 3T3 cells (27, 55). Here we compared the levels of apo-

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ptosis in NIH 3T3 cells expressing similar amounts of wild-type or mutant (Ala-53) eIF4E protein. Our findings that eIF4E Ala cells manifested a level of apoptosis intermediate between those of NIH 3T3 neo and eIF4E wt cells are in accord with the result of experiments with Xenopus embryos (25). In that system, overexpression of wild-type eIF4E induces mesodermal differentiation in 90% of embryos, compared with 53% for eIF4E with Ala-53. Unlike primary fibroblasts, established cell lines downregulate cell cycle inhibitors or constitutively express cell cycle promoters that give them a growth advantage over their nonestablished counterparts. The mechanism that sensitizes immortalized fibroblasts toward apoptosis is uncertain. One explanation is that along with immortality comes the inability to effectively downregulate the apoptotic pathway when growth factors are absent. For example, immortalization of B cells by Epstein-Barr virus leads to both deregulated expression of Myc and apoptotic death under growth-factor-restricted conditions (6). In immortalized cells, apoptosis under growth-factor-restricted conditions is not associated with growth arrest. It occurs in the context of continued proliferation induced by activation of growth-promoting signals such as E2F (17, 68) or Myc (2, 8). In contrast to normal fibroblasts with physiological Myc expression, which undergo growth arrest under low-serum conditions but survive, cells constitutively expressing Myc continue to proliferate under the same conditions and die in a growth-factor-dependent manner (2, 7, 8). Our findings demonstrate that overexpression of eIF4E abrogates this death response, as well as the death of growth-factor-restricted NIH 3T3 cells. It is possible, therefore, that eIF4E interdicts a stage in the apoptotic pathway downstream of Myc-dependent events. Myc is a transcriptional activator of eIF4E (52). Thus, Myc not only activates pathways leading to cell cycle transit and apoptosis but also provides negative feedback via eIF4E to the apoptotic pathway. This feedback survival loop, however, is not sufficient to rescue cells overexpressing Myc under growthfactor-restricted conditions. The critical importance of external cytokines for survival of Myc-overexpressing cells might be due to the role of growth factors in activating eIF4E function by phosphorylation of its negative regulator, 4E-BP1 (13, 15, 30), as well as eIF4E itself (10, 33, 47). In addition, we have recently found that while the level of eIF4E is upregulated by constitutively expressed Myc, it was significantly decreased following the shift of REF/Myc fibroblasts to serum-restricted medium (49). Thus, whether REF/Myc will undergo apoptosis is apparently regulated both by the balance between eIF4E and Myc protein levels and by posttranscriptionally controlled growth-factor-dependent events. Apoptosis frequently results when cells are driven to cycle by a proliferative signal in the context of negative growth regulators such as serum restriction or cytostatic agents (reviewed in reference 7). Our findings indicate that in the case of eIF4E, a proliferative signal (i.e., eIF4E overexpression) accompanying a negative growth regulator (growth factor withdrawal) can actually prevent apoptosis. There are at least three explanations for this phenomenon. First, the survival signal induced by eIF4E may result from cell cycle transit itself. Second, the survival signal might be an integral part of the reactions activating the proliferative machinery, so that actual cell cycle transit is not required for survival (21). Third, activated eIF4E might stimulate proliferation and survival through distinct regulatory pathways. Our current observation that eIF4E inhibits apoptosis in nonproliferating, serum-deprived cells indicates that the antiapoptotic effect of eIF4E is unlikely to depend on cell cycle

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transit. Also in accord with a salvage pathway independent of cell cycle progression is the behavior of fibroblasts expressing upregulated Ras. The ability of eIF4E to promote cell cycling and malignant transformation depends on Ras-mediated pathways (28, 29, 47). In agreement with the observations of Wyllie and his colleagues (70), our present results demonstrate that, unlike that of Myc, overexpression of Ras is associated only with increased proliferation. These data indicate that the antiapoptotic effect of eIF4E is not directly linked either to its proliferative signaling mediated by Ras or to cell cycle transit. The present data do not resolve whether eIF4E-mediated survival signaling results from a Ras-independent proliferative pathway metabolite that by itself is not sufficient to promote cell cycle transit or whether the eIF4E-dependent survival and proliferative pathways are completely independent. The involvement of translational control in apoptosis regulation provides further insights into possible mechanisms of growth factor survival signaling. While we found that both PDGF and IGF-1 suppress Myc-induced apoptosis in fibroblasts, IGF-1 has been previously shown to act posttranslationally, whereas PDGF required de novo protein synthesis to suppress apoptosis (14). PDGF and IGF-1 signaling pathways differ. PDGF leads to induction of immediate-early growth genes such as c-myc, whereas IGF-1 does not. On the basis of this, we propose a working model for PDGF survival signaling which includes the activation of pathways (such as those downstream of Myc) that lead to both proliferation and apoptosis. The choice between cell division and death is determined by a growth-factor-activated translationally controlled event, perhaps mediated by eIF4E, which in turn stimulates two discrete pathways. One leads to cell cycle progression, and the other blocks apoptosis. IGF-1 survival signaling, which is translationally independent, operates through a second salvage pathway that apparently functions in tandem with translational control to promote viability. Both Myc (52) and Ras (11) are upstream activators of eIF4E. However, coexpression of Myc and Ras was unable to salvage growth-factor-restricted fibroblasts. This implies that if growth factors mediate survival by activating eIF4E, it is through pathways distinct from those involving Myc and Ras. One candidate pathway includes p70s6K, which is activated by both PDGF and IGF-1. This pathway leads to the phosphorylation and inactivation of the eIF4E-inhibitory binding protein, 4E-BP1, in a mitogen-activated protein kinase/Ras-independent manner (13). In summary, our results provide evidence that eIF4E suppresses Myc-induced apoptosis and replaces the function of serum survival factors in fibroblasts. It is currently unclear what downstream effectors mediate the antiapoptotic function of eIF4E. Our findings point away from some of the known members of the Bcl-2 protein family. They suggest that candidate effectors are translationally regulated by eIF4E and inhibit the apoptotic pathway upstream of the execution apparatus. Myc has a dual role in regulating cellular fate, activating pathways leading to proliferation and death. Growth factors block the death pathway. Our data suggest that eIF4E may participate in some of these salvage pathways downstream of the growth factor-receptor interaction. Our data also provide an additional explanation for how Myc cooperates with eIF4E in the neoplastic transformation of primary rodent fibroblasts (29). The observed synergism can be, at least in part, related to the antiapoptotic function of eIF4E. Since a variety of tumor cell lines exhibit elevated levels of eIF4E mRNA expression (36, 48, 49), activation of the eIF4E-mediated survival pathway, in addition to its growth-promoting function, may be of critical

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importance in the development of neoplasia as well as other proliferative disorders. ACKNOWLEDGMENTS This work was supported by a grant from the NIH SCOR in Acute Lung Injury (1-P50 HL50152-01) to P. Bitterman, V. Polunovsky, and J. White. We thank T. Behrens for valuable advice and Bcl reagents. REFERENCES 1. Almasan, A., Y. X. Yin, R. E. Kelly, E. Y. H. P. Lee, A. Bradley, W. W. Li, J. R. Bertino, and G. M. Wahl. 1995. Deficiency of retinoblastoma protein leads to inappropriate S-phase entry, activation of E2F-responsive genes, and apoptosis. Proc. Natl. Acad. Sci. USA 92:5436–5440. 2. Askew, D. S., R. A. Ashmun, B. C. Simmons, and J. L. Cleveland. 1991. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene 6:1915–1922. 3. Boise, L. H., M. Gonzalez-Garcia, C. E. Postema, L. Ding, T. Lindsten, L. A. Turka, X. Mao, B. Nunez, and C. B. Thompson. 1993. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74:597–608. 4. Bommer, U.-A., A. Lazaris-Karatzas, A. DeBenedetti, P. Nurnberg, R. Benndorf, H. Bielka, and N. Sonenberg. 1994. Translational regulation of the mammalian growth-related protein p23: involvement of eIF4E. Cell. Mol. Biol. Res. 40:633–641. 5. Brooks, R. F. 1977. Continuous protein synthesis is required to maintain the probability of entry into S phase. Cell 12:311–317. 6. Cherney, B. W., K. Bhatia, and G. Tosato. 1994. A role for deregulated c-Myc expression in apoptosis of Esptein-Bar virus-immortalized B cells. Proc. Natl. Acad. Sci. USA 91:12967–12971. 7. Evan, G. I., L. Brown, M. Whyte, and E. Harrington. 1995. Apoptosis and the cell cycle. Curr. Opin. Cell. Biol. 7:825–834. 8. Evan, G. I., A. H. Wyllie, C. Gilbert, T. D. Littlewood, H. Land, M. Brooks, C. M. Waters, L. Z. Penn, and D. C. Hancock. 1992. Induction of apoptosis in fibroblasts by c-myc protein. Cell 69:119–128. 9. Fanidi, A., E. A. Harrington, and G. I. Evan. 1992. Cooperative interaction between c-myc in inhibited and bcl-2 protooncogenes. Nature (London) 359:554–556. 10. Frederickson, R. M., K. S. Montine, and N. Sonenberg. 1991. Phosphorylation of eukaryotic translation initiation factor 4E is increased in Src-transformed cell lines. Mol. Cell. Biol. 11:2896–2900. 11. Frederickson, R. M., W. E. Mushynski, and N. Sonenberg. 1992. Phosphorylation of translation initiation factor eIF4E is induced in ras-dependent manner during nerve growth factor-mediated PC12 cell differentiation. Mol. Cell. Biol. 12:1239–1247. 12. Gavrielli, Y., Y. Sherman, and S. A. Ben-Sasson. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 19:493–501. 13. Graves, L. E., K. E. Bornfeldt, G. M. Argast, E. G. Krebs, X. Kong, T.-A. Lin, and J. C. Lawrence, Jr. 1995. cAMP-and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-1 in aortic smooth muscle cells. Proc. Natl. Acad. Sci. USA 92:7222–7226. 14. Harrington, E. A., M. R. Bennett, A. Fanidi, and G. I. Evan. 1994. c-Mycinduced apoptosis in fibroblasts is inhibited by specific cytokines. EMBO J. 13:3286–3295. 15. Haystead, T. A. J., C. M. M. Haystead, C. Hu, T.-A. Lin, and J. C. Lawrence, Jr. 1994. Phosphorylation of PHAS-1 by mitogen-activated protein (MAP) kinase. J. Biol. Chem. 269:23185–23191. 16. Hernanedes, G., and J. M. Sierra. 1995. Translation initiation factor eIF4E from drosophila-cDNA sequence and expression of the gene. Biochim. Biophys. Acta 1261:427–431. 17. Hiebert, S. W., G. Packham, D. K. Strom, R. Haffner, M. Oren, G. Zambetti, and J. L. Cleveland. 1995. E2F-1:DP-1 induces p53 and overrides survival factors to trigger apoptosis. Mol. Cell. Biol. 15:6864–6874. 18. Hoang, A. T., K. J. Cohen, J. F. Barrett, D. A. Bergstrom, and V. C. Dang. 1994. Participation of cyclin A in Myc-induced apoptosis. Proc. Natl. Acad. Sci. USA 91:6875–6879. 19. Hockenbery, D. M. 1995. bcl-2, a novel regulator of cell death. Bioessays 17:631–638. 20. Ishizaki, Y., L. Cheng, A. W. Mudge, and M. C. Raff. 1995. Programmed cell death by default in embryonic cells, fibroblasts and cancer cells. Mol. Biol. Cell 6:1443–1458. 21. Jewell, A. P., P. M. Lydyard, C. P. Worman, F. J. Giles, and A. H. Goldstone. 1995. Growth factors can protect B-chronic lymphocytic leukaemia cells against programmed cell death without stimulating proliferation. Leuk. Lymphoma 18:159–162. 22. Joshi, B., A.-L. Cai, B. D. Keiper, W. B. Minich, R. Mendez, C. M. Beach, J. Stepinski, R. Stolarski, E. Darzynkiewicz, and R. E. Rhoads. 1995. Phosphorylation of eukaryotic protein synthesis initiation factor 4E at Ser-209.

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