Abstract
Proliferation and cell cycle progression in response to growth factors require de novo protein synthesis. It has been proposed that binding of the eukaryotic translation initiation factor 4E (eIF-4E) to the inhibitory protein 4BP-1 blocks translation by preventing access of eIF-4G to the 5′ cap of the mRNA. The signal for translation initiation is thought to involve phosphorylation of 4BP-1, which causes it to dissociate from eIF-4E and allows eIF-4G to localize to the 5′ cap. It has been suggested that the ability of the macrolide antibiotic rapamycin to inhibit 4BP-1 phosphorylation is responsible for the potent antiproliferative property of this drug. We now show that rapamycin-resistant cells exhibited normal proliferation despite dephosphorylation of 4BP-1 that allows it to bind to eIF-4E. Moreover, despite rapamycin-induced dephosphorylation of 4BP-1, eIF-4E–eIF-4G complexes (eIF-4F) were still detected. In contrast, amino acid withdrawal, which caused a similar degree of 4BP-1 dephosphorylation, resulted in dissociation of the eIF-4E–eIF-4G complex. Thus, 4BP-1 dephosphorylation is not equivalent to eIF-4E inactivation and does not explain the antiproliferative property of rapamycin.
Cellular proliferation involves translation of specific mRNAs encoding proteins required for transition from the G1 phase to the S phase of the cell cycle (6, 30). Both proliferative and antiproliferative stimuli (26, 35) can regulate translation initiation, which is the rate-limiting step in de novo protein synthesis. Eukaryotic initiation factor 4E (eIF-4E) has been proposed as the critical regulator of translation (26). eIF-4E binds to the 7-methyl GTP (m7-GTP) cap on the 5′ untranslated region of all cytoplasmic eukaryotic mRNAs and recruits the 40S ribosomal complex. This complex comprises eight proteins, including eIF-4A (RNA helicase), eIF-4B (RNA binding protein), and eIF-4G, a scaffolding protein that directly interacts with eIF-4E and is believed to unwind the secondary structure of the 5′ untranslated region, allowing efficient translation initiation (26, 33).
4BP-1 (or PHAS-I) has been identified as an important inhibitor of eIF-4E (23, 33). 4BP-1 is thought to inhibit translation initiation by binding to eIF-4E (which is continuously bound to the 5′ cap) and preventing its association with eIF-4G (14). Phosphorylation of 4BP-1 causes it to dissociate from eIF-4E, thereby allowing translation initiation to proceed (7, 11, 23). Overexpression of 4BPs (4BP-1 and 4BP-2) in cells transformed by either eIF-4E or v-src causes significant reversion of the transformed phenotype, suggesting that members of the 4BP family are negative regulators of cell growth (43).
The mTOR/FRAP/RAFT1 (4, 17, 44) protein has been shown to regulate phosphorylation of 4BP-1 (7, 15, 23, 33) and p70s6k (5). Rapamycin bound to its cytosolic receptor, the FK506 binding protein (FKBP12) (45), inhibits the kinase activity of mTOR/FRAP/RAFT1, resulting in dephosphorylation of 4BP-1, increased 4BP-1–eIF-4E complex formation, and, presumably, inhibition of translation initiation (7, 11, 13, 23, 28, 33, 46). It has been proposed that inactivation of eIF-4E via 4BP-1 is the mechanism whereby rapamycin inhibits G1-to-S-phase progression (7). However, disruption of the gene encoding 4BP-1 (PHAS-I) in mice does not cause rapamycin resistance, and fibroblasts derived from these mice exhibit normal protein synthesis and growth (2). Furthermore, 4BP-1 may not play a significant role in rapamycin’s antiproliferative effects, as suggested by the findings that rapamycin does not prevent the early effects of serum-induced protein translation, polysome formation (34), eIF-4E phosphorylation, or the recruitment of eIF-4E into the eIF-4F complex (29).
mTOR has also been implicated in the pathway(s) mediating nutrient sensing through dephosphorylation of both p70s6k (3, 16, 48) and 4BP-1 (16, 48). For example, amino acid withdrawal in Chinese Hamster Ovary (CHO) cells causes p70s6k dephosphorylation and kinase inhibition, 4BP-1 dephosphorylation, increased 4BP-1–eIF-4E association, and reduced eIF-4E–eIF-4G complex formation (16, 48). Rapamycin inhibits the ability of amino acids to induce the release of 4BP-1 from eIF-4E and inhibits the complex formation of eIF-4E–eIF-4G (48). Therefore, the effect of rapamycin on eIF-4E–eIF-4G complex formation appears to be related to the method of stimulation; rapamycin does not inhibit serum-induced eIF-4E–eIF-4G complex formation (29), whereas it does inhibit amino acid-induced complex formation (48).
In the present study, we examined the effects of rapamycin on modulators of protein translation in four different cell lines. We found that in CHO cells, BC3H1 cells, and two rapamycin-resistant (RR) cell lines, i.e., (i) RR cells generated from BC3H1 cells and (ii) murine erythroleukemia cells (MELC), rapamycin caused dephosphorylation of 4BP-1 and increased association of 4BP-1 and eIF-4E without causing eIF-4E–eIF-4G dissociation. These results suggest that rapamycin does not cause cell cycle arrest through inhibition of the eIF-4E–eIF-4G complex formation.
MATERIALS AND METHODS
Cell culture.
BC3H1 and RR cells were grown in Dulbecco’s modified essential medium (DMEM) with the addition of 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The medium was changed every 48 to 72 h. Rapamycin was added directly to the medium. The cells were cultured for multiple passages in the presence of high concentrations of rapamycin (0.1 to 1 μM) followed by dilutional cloning. Two independent clones were utilized; RR-1 clones were grown in 1 μM rapamycin, and RR-3 clones were grown in 0.1 μM rapamycin. In all studies, rapamycin was removed 1 week prior to the experiment. MELC were grown in minimal essential medium-α (MEM-α) supplemented with 10% FBS (inactivated) and 1% penicillin-streptomycin (38). CHO cells (obtained from the American Type Culture Collection) were grown in Ham’s F-12 medium supplemented with 10% FBS and 1% penicillin-streptomycin. Deprivation and restoration of amino acids were performed as previously described (48).
Cell proliferation assays.
BC3H1, RR-1, and RR-3 cells (25 × 104) were grown in DMEM plus 20% FBS (in triplicate). Rapamycin (100 nM) was added directly to the medium. After 48 h, the cell number was determined with a Coulter Counter. MELC (10 × 104) were grown in MEM plus 10% FBS; rapamycin (0.2 to 1 μM) or a vehicle was added directly to the medium. After 4 days, the cells were counted with a Coulter Counter.
[3H]leucine incorporation.
BC3H1 and RR-1 cells (104) were plated in triplicate in 12-well dishes. After 48 h, rapamycin (1 μM) was added to the appropriate wells, and cells were pulsed with 1 μCi of [3H]leucine per well. [3H]leucine incorporation into protein was determined by precipitation with trichloroacetic acid. Experiments were repeated three times.
Fluorescence-activated cell sorter analysis.
BC3H1 and RR-1 cells were placed in DMEM plus 1% FBS for 24 h. The cells were then stimulated with DMEM plus 20% FBS and treated with either rapamycin (100 nM) or the vehicle. The cells were washed and harvested after 24 h and labeled with propidium iodide solution-RNase for 1 h. The cells were analyzed on a fluorescence-activated cell sorter, with a minimum of 15,000 cells counted.
Western blot analyses.
To detect 4BP-1, eIF-4E, and eIF-4G, lysates were prepared as previously described (23). Parental BC3H1 cells and RR cells, MELC, and CHO cells were grown in DMEM plus 20% FBS, MEM plus 10% FBS (inactivated), and Ham’s F-12 medium plus 10% FBS, respectively. Asynchronously dividing cells were treated with either the vehicle or rapamycin for 30 min, and cellular lysates were prepared. Protein was measured with the Bradford reagent, size fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose. Membranes were incubated as previously described (23) and visualized by enhanced chemiluminescence. For 4BP-1–eIF-4E complex experiments, cell extracts (130 μg) were incubated with m7-GTP–Sepharose in duplicate for 1 h at 4°C. The resin was washed and samples were size fractionated on SDS–8% (eIF-4G) or –13% (eIF-4E and 4BP-1) polyacrylamide gels, transferred to nitrocellulose, and blotted with eIF-4G (29), eIF-4E (Transduction Laboratories), or 4BP-1 (PHAS-I) (23) antibodies. For immunoprecipitation, cell extracts (100 μg) were incubated with 2 μl of anti-eIF-4G antibody (49) in 500 μl of lysis buffer, captured with protein A-Sepharose, washed and eluted with Laemmli buffer, and size fractionated on SDS–11.5% polyacrylamide gels.
RESULTS AND DISCUSSION
Proliferation and cell cycle progression in RR cells.
RR and BC3H1 cells were selected on the basis of normal growth following multiple passages in the presence of high concentrations of rapamycin (either 100 or 1,000 nM). Proliferating RR and parental cells exhibited similar morphologies (Fig. 1A). Two independent clonal RR cell lines (RR-1 and RR-3) exhibited no growth arrest (Fig. 1B) and no decrease in viability (as assessed by trypan blue exclusion) in the presence of rapamycin. Rapamycin induced a delay in transition from G1 to S phase in the parental cells but not in the RR cells (Fig. 1C).
FIG. 1.
Growth characteristics of RR cells. (A) The morphologies and sizes of proliferating parental (BC3H1) and RR-1 cells are similar. (B) Rapamycin inhibited cell growth in the parental cells but not in two RR cell lines, RR-1 and RR-3. Hatched bars show the numbers of cells that were plated; cell number was determined after 48 h. Growth was significantly inhibited for rapamycin-treated cells (100 nM, black bars) compared to untreated BC3H1 cells (white bar). Data are averages of triplicate samples + standard deviations. (C) Flow cytometry of parental and RR-1 cells. Cells were serum starved in DMEM plus 1% FBS for 24 h, followed by stimulation with 20% FBS. Cells were harvested after 24 h and stained with propidium iodide. Analysis was based upon results from a minimum of 15,000 cells. Parental cells treated with rapamycin (100 nM) arrested in G1/S; RR-1 cells showed no rapamycin-induced inhibition of cell cycle progression.
Rapamycin inhibits activation of cyclin-dependent kinases (CDK) by preventing mitogen-induced down-regulation of the CDK inhibitor p27kip1 (31). p27kip1 protein regulation has been shown to be controlled at the translational level (18) and via ubiquitin-dependent degradation (32). Rapamycin resistance in RR cells results from a deficiency in p27kip1 due to increased degradation of the protein via a ubiquitin-independent pathway (25). RR cells have constitutively low p27kip1 levels, and unlike the case with parental cells, p27kip1 levels are not regulated in response to mitogens or rapamycin (25). In response to serum withdrawal, RR cells undergo apoptosis because of their inability to extinguish hyperphosphorylation of retinoblastoma protein (pRb) (25).
Inhibition of 4BP-1 phosphorylation.
The ability of RR cells to proliferate in the presence of high concentrations of rapamycin provided the opportunity to determine whether inactivation of proteins thought to be required for translation interferes with cell cycle progression. Rapamycin has been previously demonstrated to minimally inhibit protein synthesis, if at all (11, 36). In both proliferating BC3H1 and RR-1 cells, rapamycin (1 μM) demonstrated no significant effect on [3H]leucine incorporation (Fig. 2A).
FIG. 2.
Generalized protein synthesis is unaffected by rapamycin, but 4BP-1 is inhibited in parental and RR cells. (A) Parental BC3H1 and RR-1 cells (104) were cultured in DMEM plus 20% FBS for 48 h in 12-well dishes; 1 μM rapamycin (black bars) or vehicle (white bars) was added to the appropriate wells when the cells were pulsed with [3H]leucine (1 μCi). Incorporation of [3H]leucine was measured by precipitation with trichloroacetic acid. Rapamycin had no significant effect on protein synthesis. Data are representative of three experiments. (B) Parental BC3H1, RR-1, and RR-3 cells were cultured in 20% FBS; 100 nM rapamycin or vehicle was added to the cultures, and lysates were prepared after 45 min. Cellular lysates (100 μg) were analyzed by immunoblotting with an anti-4BP-1 (anti-PHAS-I) antiserum. α, β, and γ are arbitrary designations of bands representing the phosphorylated forms of 4BP-1 (23).
Rapamycin inhibited phosphorylation of both p70s6k (data not shown and reference 25) and 4BP-1 (Fig. 2B) in proliferating RR cells. The inhibition of both p70s6k and 4BP-1 phosphorylation in wild-type and RR cells was observed more than 48 h after removal of rapamycin from the culture medium (data not shown). Rapamycin is a potent inhibitor of cell growth; therefore, it has been difficult to determine whether the drug specifically inhibits translation or, conversely, whether the primary effect of rapamycin is to inhibit cell cycle progression (e.g., by up-regulating p27kip1 [25, 31]). However, the ability of rapamycin to inhibit 4BP-1 (PHAS-1) phosphorylation and eIF-4E function has been interpreted as indicating that inhibition of translation is the mechanism underlying the growth-inhibitory properties of rapamycin (7). The rapamycin-induced inhibition of 4BP-1 in proliferating RR cells uniquely demonstrates that proliferation can proceed despite dephosphorylation of 4BP-1.
4BP-1–eIF-4E interaction.
Overexpression of eIF-4E increases the translation of mRNAs containing extensive secondary structures, including cyclin D1 (40, 41), ornithine decarboxylase (42), and c-myc (9), and causes transformation of fibroblasts (10, 22, 39). Immunoblot analyses showed that eIF-4E protein levels were equivalent in the parental and RR cells (Fig. 3A), indicating that the stoichiometry between 4BP-1 (Fig. 2B) and eIF-4E was unchanged in the RR cells. eIF-4E is constitutively bound to the 5′ cap, and its activity is regulated by the binding of the inhibitory protein 4BP-1 (23, 33). To determine whether 4BP-1 was bound to eIF-4E in RR cells, an affinity resin containing the 5′ cap homolog m7-GTP was used as previously described (23). In these experiments dephosphorylation of 4BP-1 caused 4BP-1 to bind to eIF-4E, which in turn was bound to the m7-GTP resin. RR cells cultured with rapamycin were able to proliferate despite persistent inactivation of eIF-4E by 4BP-1 (Fig. 3B). No significant differences in the amount of 4BP-1 binding to eIF-4E were seen in BC3H1 and RR cells treated with rapamycin.
FIG. 3.
Rapamycin increases complex formation between 4BP-1 and eIF-4E in parental and RR cells. (A) Cell extracts (100 μg) were analyzed by immunoblotting with anti-eIF-4E antibody. eIF-4E protein levels were equivalent in parental BC3H1 and RR cells. (B) Binding of 4BP-1 to eIF-4E (already bound to m7-GTP–Sepharose resin) was determined. m7-GTP–Sepharose was incubated with 130 μg of cellular extract for 1 h at 4°C. The resin was washed, size fractionated by SDS-PAGE, transferred to nitrocellulose, and blotted with anti-4BP-1 or anti-eIF-4E antibody.
Like RR cells, MELC exhibited no growth arrest after 4 days of rapamycin treatment (Fig. 4A). Rapamycin also inhibits p70s6k in MELC, as previously reported (8). In proliferating MELC, rapamycin (0.2 μM) treatment for 1 h inhibited 4BP-1 phosphorylation (Fig. 4B) and increased the complex formation between 4BP-1 and eIF-4E as assessed by m7-GTP binding (Fig. 4C). Rapamycin had no effect on eIF-4E protein levels in MELC (data not shown).
FIG. 4.
MELC are rapamycin resistant and demonstrate rapamycin-induced 4BP-1 dephosphorylation and increased 4BP-1–eIF-4E complex formation. (A) MELC (10 × 104 cells; hatched bar) were treated with either a vehicle (white bar) or increasing concentrations of rapamycin (black bars [numbers are micromolar concentrations]). Cells were counted at 4 days. These data are representative of two experiments. (B) Cell extracts were analyzed by immunoblotting with an anti-4BP-1 antibody. Rapamycin (0.2 μM) treatment for 1 h induced dephosphorylation of 4BP-1. α, β, and γ are arbitrary designations of bands representing the phosphorylated forms of 4BP-1 (23). Data are representative of three experiments. (C) Binding of 4BP-1 to eIF-4E (bound to m7-GTP resin) was determined in the presence or absence of rapamycin (0.2 μM). Cellular extracts were incubated with m7-GTP resin, size fractionated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-4BP-1 and anti-eIF-4E antibodies. Rapamycin increased the binding of 4BP-1 to eIF-4E.
Rapamycin does not affect eIF-4E–eIF-4G complex formation.
The binding of eIF-4G or 4BP-1 to eIF-4E is believed to be mutually exclusive (14, 36). Therefore, the increased association of eIF-4E and 4BP-1 would theoretically reduce the association of eIF-4E and eIF-4G (7, 14, 24, 47). Several groups have shown that increased eIF-4E–eIF-4G association was correlated with decreased 4BP-1–eIF-4E complex formation (12, 21). Moreover, rapamycin prevented amino acid-induced eIF-4E–eIF-4G complex formation in amino acid-depleted CHO cells (48). However, conflicting data have also been reported with regard to the effects of rapamycin on eIF-4E–eIF-4G interactions (20, 29). Morley and McKendrick demonstrated with NIH 3T3 cells that rapamycin increased the association of 4BP-1 and eIF-4E on m7-GTP resin but that rapamycin did not prevent serum-induced eIF-4E–eIF-4G interaction as assessed by immunoprecipitation assays (29). Moreover, they demonstrated that rapamycin does not inhibit the recruitment of eIF-4E to the ribosome and that prolonged incubation (for 20 h) causes only a 30 to 40% reduction in the amount of eIF-4E associated with eIF-4G (29). Furthermore, Beretta et al. (1) have shown a lack of temporal correlation between 4BP-1 dephosphorylation and inhibition of in vitro cap-dependent translation. Morley and McKendrick (29) and others (1) have suggested that these data may reflect a low rate of release of 4BP-1 from eIF-4E, such that eIF-4E is able to associate with 4BP-1 only after release from eIF-4G.
In CHO cells, rapamycin did not prevent the association of eIF-4E and eIF-4G as assessed by immunoprecipitation and binding to m7-GTP resin (Fig. 5A). However, as previously described (48), amino acid deprivation for 1 h did significantly reduce the association of eIF-4E and eIF-4G. Rapamycin (1 μM) did not prevent serum-induced association of eIF-4E and eIF-4G. Morley and McKendrick (29) suggested that there might be a population of eIF-4E that is inaccessible to dephosphorylated 4BP-1 and thus dissociation from eIF-4G. However, our findings that amino acid withdrawal rapidly induced dissociation of the eIF-4E–eIF-4G complex in cells in which serum withdrawal did not induce the same dissociation suggest that compartmentalization may not explain the lack of effect of rapamycin on this complex in both CHO cells and BC3H1 or RR cells.
FIG. 5.
Rapamycin does not prevent serum-induced association of eIF-4E–eIF-4G in CHO, BC3H1, and RR cells. (A) CHO cells were grown in DMEM plus 10% FBS and treated with either a vehicle or rapamycin for 24 h. In parallel experiments, CHO cells were placed in Ham’s F-12 medium without serum for 16 h. Cells were washed and amino acid deprived in Earle’s salt solution for 1 h. Cells were stimulated with Ham’s F-12 medium plus 10% FBS following pretreatment with either a vehicle or rapamycin (1 μM). Cell lysates were prepared as described in Materials and Methods. Cellular extracts (100 μg) were incubated with m7-GTP resin at 4°C in duplicate, washed, size fractionated on an SDS–8% polyacrylamide gel, transferred to nitrocellulose, and immunoblotted with anti-eIF-4G antibody (the nitrocellulose was also incubated with anti-eIF-4E antibody to demonstrate equal uptake on the resin [data not shown]). Cellular extracts (100 μg) were immunoprecipitated with anti-eIF-4G antibody, bound with protein A-Sepharose beads, size fractionated on an SDS–11.5% polyacrylamide gel, transferred to nitrocellulose, and immunoblotted with either anti-eIF-4E or anti-eIF-4G antibodies. Amino acid (AA) withdrawal significantly inhibited eIF-4E–eIF-4G complex formation. Rapamycin had no effect on serum-stimulated eIF-4E–eIF-4G association. Data are representative of three similar experiments. (B) BC3H1 and RR-1 cells were grown in DMEM plus 20% FBS. Experiments were performed as described above (in duplicate) to determine the binding of eIF-4G and 4BP-1 to eIF-4E bound to m7-GTP–Sepharose resin. Size fractionation was performed by SDS-PAGE with either 8% (eIF-4G) or 13% (4BP-1) polyacrylamide. Rapamycin (1 μM) was added to the medium 24 h prior to cell lysis. Rapamycin and serum withdrawal (cells maintained in DMEM alone) for 1 h caused increased association of 4BP-1 with the m7-GTP resin in BC3H1 and RR cells. However, complex formation between eIF-4E and eIF-4G persisted despite rapamycin (1 μM) treatment for 24 h.
In BC3H1 and RR cells, rapamycin (1 μM for 24 h) caused increased association of 4BP-1 with the m7-GTP resin but failed to inhibit the serum-induced association of eIF-4E and eIF-4G as assessed by binding to the m7-GTP (Fig. 5B) and by coimmunoprecipitation (Fig. 6). In RR cells exposed to rapamycin for more than 1 week, eIF-4G–eIF-4E association persisted despite increased 4BP-1 association with eIF-4E (data not shown), indicating that even prolonged exposure to rapamycin and 4BP-1 dephosphorylation does not induce eIF-4E–eIF-4G dissociation. Amino acid withdrawal in BC3H1 and RR cells caused increased association of 4BP-1 and eIF-4E on m7-GTP resin and markedly reduced eIF-4E–eIF-4G complex formation (Fig. 6). Rapamycin inhibited the amino acid-induced eIF-4E–eIF-4G complex formation in nutrient-deprived BC3H1 cells; however, rapamycin did not inhibit the serum-induced eIF-4E–eIF-4G complex formation in BC3H1 and RR cells (Fig. 6). Therefore, although growth factor (serum) withdrawal can lead to dephosphorylation of 4BP-1 and increased 4BP-1–eIF-4E association, in the absence of amino acid withdrawal, it is insufficient to cause eIF-4E–eIF-4G complex dissociation.
FIG. 6.
Differential regulation of the eIF-4E–eIF-4G complex by rapamycin and amino acid withdrawal in BC3H1 and RR-1 cells. BC3H1 and RR-1 cells were cultured in DMEM plus 20% FBS and treated with rapamycin (1 μM). In amino acid (AA) withdrawal experiments, cells were placed in Earle’s balanced salt solution for 1 h. Cells were pretreated with rapamycin (1 μM) for 15 minutes and then stimulated for 30 min either with amino acids or with 20% FBS plus amino acids. Cellular extracts were prepared. eIF-4G and eIF-4E were coimmunoprecipitated with an anti-eIF-4G antibody and immunoblotted with either anti-eIF-4G or anti-eIF-4E antibody. In addition, the same extracts were used in experiments performed as described above (in duplicate) to determine the binding of 4BP-1 to eIF-4E (already bound to m7-GTP–Sepharose resin).
Conclusions.
The mechanism(s) by which rapamycin inhibits proliferation and induces G1/S arrest have not been fully elucidated. Rapamycin induces a G1-to-S-phase transition arrest and inhibits a mitogen-activated signaling pathway that involves mTOR, p70s6k, p27kip1, 4BP-1, eIF-4E, cell cycle kinases, and retinoblastoma protein (5, 7, 11, 23, 28, 33). However, the immediate downstream targets of mTOR and the significance of rapamycin’s inhibition of p70s6k, 4BP-1, and eIF-4E remain uncertain. The observations by several groups that the inhibition of mTOR, p70s6k, and 4BP-1 phosphorylation by rapamycin were coupled to growth arrest and to inactivation of eIF-4E led to the hypothesis that rapamycin’s antiproliferative properties are mediated via translational control (7, 13, 19, 37). In the present study, we used two RR muscle cell lines (RR-1 and RR-3) and a third hematopoietic cell line (MELC) that is also resistant to rapamycin and confirmed our findings with rapamycin-sensitive CHO cells. This strategy allowed us to address the question of whether cell cycle progression and translation can proceed despite dephosphorylation of 4BP-1 and inactivation of p70s6k by rapamycin. We showed that rapamycin-mediated dephosphorylation and increased binding of eIF-4E to 4BP-1 are not required for rapamycin’s antiproliferative effects. Rapamycin does not cause the dissociation of the eIF-4E–eIF-4G complex in serum-stimulated cells; therefore, inhibition of protein translation does not appear to be the mechanism through which rapamycin exerts its antiproliferative effects. Rapamycin caused no significant reduction in protein synthesis (Fig. 2A) (11, 36), in contrast to amino acid withdrawal, which has a more profound effect on generalized protein synthesis (16). Although amino acid withdrawal and rapamycin cause similar degrees of dephosphorylation of p70s6k and 4BP-1, presumably through inhibition of mTOR activity, they do not have similar effects on the association of eIF-4E and eIF-4G. This suggests that the mTOR pathway, which is regulated by amino acids and rapamycin, may play a greater role in p70s6k inhibition and 4BP-1 phosphorylation than in eIF-4E–eIF-4G complex regulation.
The ability of rapamycin to inhibit cellular proliferation may have important applications in the treatment of disorders such as accelerated arteriopathy that occurs in transplanted hearts and restenosis following the placement of coronary stents (27). The present study indicates that the antiproliferative properties of rapamycin are probably not mediated by inhibition of protein translation via inactivation of p70s6k or eIF-4E. While it appears that rapamycin does not inhibit cell growth via translational control, the CDK inhibitors, in particular p27kip1, remain attractive candidates for mediators of the drug’s important antiproliferative properties.
ACKNOWLEDGMENTS
We thank J. Blenis for anti-p70s6k antibody, J. Lawrence for anti-PHAS-I (anti-4BP-1) and anti-eIF-4E antibodies, S. Morley and R. Rhoads for anti-eIF-4G antibodies, V. Richon for MELC and for data on the effects of rapamycin on MELC growth, and J. Hurwitz and D. Cobrinik for critical reading of the manuscript.
This work was supported by the NIH, MDA, and AHA (A.R.M.) and the Richard and Lynne Kaiser Family Foundation and by a grant from the Johnson and Johnson Focused Giving Program. S.O.M. is a recipient of an American Heart Association Clinician Scientist Award and the NY Academy of Medicine Glorney-Raisbeck Fellowship.
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