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Originally published In Press as doi:10.1074/jbc.M110782200 on February 14, 2002

J. Biol. Chem., Vol. 277, Issue 16, 13907-13917, April 19, 2002
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4E-binding Proteins, the Suppressors of Eukaryotic Initiation Factor 4E, Are Down-regulated in Cells with Acquired or Intrinsic Resistance to Rapamycin*

Michael B. Dilling, Glen S. Germain, Lorina Dudkin, Arun L. Jayaraman, Xiongwen Zhang, Franklin C. Harwood, and Peter J. HoughtonDagger

From the Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-2794

Received for publication, November 9, 2001, and in revised form, December 26, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To determine whether inhibition of either the ribosomal p70 S6 kinase or eukaryotic initiation factor (eIF) 4E pathways downstream of the mammalian target of rapamycin, mTOR, contributes to rapamycin-induced growth arrest, clones of Rh30 rhabdomyosarcoma cells were selected for rapamycin resistance. Expression of c-Myc and anchorage-independent growth were enhanced in resistant cells. Resistance was unstable in each of three clones characterized. In resistant cells, as compared with parental cells, ~10-fold less 4E-binding protein (4E-BP) was bound to eIF4E, and total cellular 4E-BP was markedly reduced. Levels of eIF4E were unchanged. Steady-state levels of 4E-BP transcript remained unaltered, but the rate of 4E-BP synthesis was reduced in resistant cells. In cells that reverted to rapamycin sensitivity, levels of total 4E-BP returned to those of parental cells. Compared with parental cells, resistant clones had either similar or lower levels and activity of ribosomal p70 S6 kinase, but c-Myc levels were elevated in both resistant and revertant clones. Several colon carcinoma cell lines with intrinsic rapamycin resistance were found to have low 4E-BP:eIF4E ratios. In stable clones of HCT8 carcinoma engineered to overexpress 4E-BP, rapamycin sensitivity increased markedly (>1000-fold) as 4E-BP expression increased. These results suggest that the 4E-BP:eIF4E ratio is an important determinant of rapamycin resistance and controls certain aspects of the malignant phenotype.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Rapamycin is a macrocyclic lactone antibiotic that potently inhibits the activation of T cells and the growth of many malignant cells in culture (1-5). Rapamycin first binds to a cytosolic immunophilin, FKBP-12; this complex then binds to the target of rapamycin (TOR)1 and inhibits its kinase function (6-9). The mammalian target of rapamycin, mTOR (also designated FRAP, RAFT1, or RAPT), has been proposed to link mitogen stimulation to protein synthesis and cell cycle progression (10). The signaling pathway downstream of mTOR bifurcates (11); hence, inhibition of mTOR signaling affects at least two separate pathways, both of which control translation of specific mRNA species. In many cell lines, exposure to rapamycin results in a relatively small (~15-20%) decrease in overall protein synthesis but causes cell cycle arrest in G1.

Inhibition of mTOR kinase activity blocks growth factor stimulation of ribosomal p70 S6 kinase and leads to hypophosphorylation of the eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) 1-3. The 4E-BP isoforms are considered to be direct substrates of mTOR kinase activity in cells (10, 12). At least two sites (Thr-37, Thr-46) on the 4E-BPs are phosphorylated by mTOR, whereas other sites (Ser-60, Thr-70, Ser-83) may be phosphorylated by mTOR (13, 14) or by other kinases (15-18). Hypophosphorylated 4E-BP family members tightly bind eIF4E, preventing its association with the multifunctional scaffolding protein eIF4G (19, 20). In serum-starved quiescent cells, stimulation with growth factor or serum leads to phosphorylation of 4E-BPs and their resultant dissociation from eIF4E, thereby facilitating assembly of the multisubunit complex (eIF4F) and efficient translation of mRNA having highly structured 5'-untranslated regions (21). Rapamycin prevents phosphorylation of 4E-BPs and prevents dissociation of the 4E-BP·eIF4E complex in growth factor-stimulated cells. The activity of mTOR is directly (22, 23) or indirectly (24) required for activation of ribosomal p70 S6 kinase through phosphorylation of its rapamycin-sensitive site (Thr-229) (25). Substitution of this residue abrogates rapamycin sensitivity. However, it is less certain whether inhibition of p70 S6 kinase activation is a crucial determinant of the action of rapamycin. Rapamycin inhibited the proliferation of embryonic stem cells in which p70 S6 kinase was disrupted (26); this finding suggests that other signaling events downstream of mTOR are crucial to growth inhibition.

Resistance to rapamycin has been extensively studied in the yeast Saccharomyces cerevisiae. Dominant mutations in the rapamycin target, TOR, that prevent its binding to the FKBP-12-rapamycin complex have been reported to cause resistance (27-29). In addition, a recessive resistance phenotype was reported to be associated with decreased expression of RBP1, a homolog of mammalian FKBP-12, or with a mutation altering Tyr-89; either of these changes decreased the formation of the rapamycin complex (30). In mammalian cells selected for resistance to rapamycin after mutagenesis, resistance is associated with a dominant phenotype consistent with a mutant mTOR (31) that has decreased binding affinity for the FKBP-rapamycin complex. Expression of a mutant mTOR (S2035I) that has reduced affinity for this complex confers marked resistance (32, 33). Rapamycin resistance is also reported to be associated with loss of the cyclin-dependent kinase inhibitor p27Kip1 (34) and to be enhanced in the cells of patients with ataxiatelangiectasia (35). Intrinsic resistance to rapamycin does not correspond to altered phosphorylation of 4E-BPs (36). This finding is consistent with our previous report that mTOR-dependent activation of p70 S6 kinase does not coincide with cellular sensitivity to rapamycin (37). Our studies also found similar levels of mTOR protein, drug uptake, and drug retention in rapamycin-sensitive and rapamycin-resistant cells. Intrinsic resistance appears to result partially from redundant signaling. For example, exogenous IGF-I completely prevents rapamycin-induced apoptosis and allows proliferation, although mTOR signaling is inhibited (33).

Resistance to rapamycin may be acquired through mutations in FKBP-12, mTOR, or p70 S6 kinase. It remains unclear, however, whether rapamycin sensitivity (growth inhibition of tumor cells) requires the inhibition of both the p70 S6 kinase and eIF4E pathways or of only one of these pathways. To answer this question, we analyzed Rh30 rhabdomyosarcoma cells selected for resistance to rapamycin. We found resistance to be unstable and to be associated with decreased intracellular 4E-BP. We observed no alterations in mTOR signaling to p70 S6 kinase or in sensitivity to inhibition by rapamycin. As expected with dysregulation of eIF4E, rapamycin-resistant cells showed increased intracellular c-Myc and significantly enhanced anchorage-independent growth. Furthermore, overexpression of 4E-BP1 dramatically sensitized colon carcinoma cells that are intrinsically resistant to rapamycin.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines-- The rhabdomyosarcoma cell lines Rh30 and Rh18 (33, 37) and the G2 glioblastoma cell line (37) were established from pediatric tumors. The human colon carcinoma cell lines CaCo2, HCT8, HCT29, and HCT116 were purchased from the American Type Culture Collection (Manassas, VA). The GC3 (38) and VRC5 colon carcinoma lines were established in this laboratory.

Cell Culture and Selection for Resistance-- Rh30 human rhabdomyosarcoma cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine (33); 300 ng/ml rapamycin was added. After 10 days, the entire monolayer was transferred to a single flask, and the rapamycin concentration was increased to 10,000 ng/ml. The cells adapted to this concentration without significant cell death and could be passaged in a 1:3 split ratio used for parental Rh30 cells. The cells were grown in 10,000 ng/ml rapamycin until colonies could be isolated. Clones were maintained in 10,000 ng/ml rapamycin until used in experiments. The rapamycin-resistant clones Rapa10K (rapamycin, 10,000 ng/ml) and C2 were selected for further study.

Development of Mutant Rapamycin-resistant Clone C4-- Rh30 cells maintained in RPMI 1640 medium supplemented with 10% FBS were exposed to 400 µg/ml mutagen ethane methylsulfonate for 16 h, and medium was then replaced. Cells were allowed to proliferate for 5 days and were then harvested; 108 cells were transferred to each of 30 Costar ACCELL peel-top flasks. Two days later, 10,000 ng/ml rapamycin in fresh medium was added to each flask. After 15 days of incubation, colonies were visible; 48 colonies were isolated by using glass cloning rings, and each was transferred to a separate well of a 24-well cluster plate containing rapamycin-free medium. The next day, 5000 ng/ml rapamycin in fresh medium was added to each well. Ten days later, when clone C4 had formed a monolayer, it was transferred to a 25-cm2 flask containing 5000 ng/ml rapamycin and was subsequently maintained in this concentration of rapamycin.

Proliferation Inhibition Assays-- Cells (105/well) were plated in triplicate on 6-well culture plates. The next day, serial dilutions of rapamycin were added to the wells; cells were then incubated for 7 days. Cells were lysed under hypotonic conditions, as described previously, and nuclei were enumerated by using a Coulter counter (4, 33).

Agar Cloning-- A solution of Bacto-Noble agar (1.2 g/100 ml; Difco) at 45 °C was added to an equal volume of 2× RPMI 1640 with 20% FBS at 37 °C to produce a 0.6% agar solution in 1× RPMI 1640 and 10% FBS. The mixture (1.5 ml) was added to each well of a six-well cluster plate (Falcon) and allowed to solidify at room temperature for ~20 min. The top layer (for cell growth) was composed of 0.3% Bacto-Noble agar made by mixing equal volumes of 1.2% agar and sterile water, both at 45 °C, to produce a 0.6% agar solution and then mixing equal volumes of the 0.6% agar solution and 2× RPMI 1640 and 20% FBS at 37 °C to yield a 0.3% agar solution in 1× RPMI 1640 and 10% FBS. Cells were added to yield a final concentration of 3000 cells/ml. One ml of the cell-containing agar was layered over the base layer in each well and allowed to solidify for 20 min at room temperature. The plates were incubated at 37 °C, 5% CO2. After 11-14 days, 0.5 ml of a solution of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma) color reagent (0.5 mg/ml in sterile PBS) was added to each well, and cells were incubated overnight at 37 °C and 5% CO2. The next day, the stained colonies were counted, and their areas were measured by using an AlphaImager 2000 Documentation & Analysis System (Alpha Innotech Corporation, San Leandro, CA).

Assay of eIF4E and 4E-BP in Tumor Cells-- Lysates were prepared as previously described (10), separated by SDS-PAGE, and immunoblotted as described previously (37). Rabbit polyclonal antibody (11208, generously provided by Nahum Sonenberg, McGill University) was used to detect 4E-BP1. A different rabbit polyclonal antibody that recognizes a carboxyl-terminal epitope common to all 4E-BP isoforms was also used (Zymed Laboratories Inc. Laboratories, South San Francisco, CA). A monoclonal antibody (Transduction Laboratories, Lexington, KY) was used to detect eIF4E.

Analysis of 4E-BP-eIF4E Binding-- A functional assay of 4E-BP1 was performed essentially as described by Gingras et al. (39). Cells were plated at a density of 3.0 × 106 cells/100-mm dish. The next day they were transferred to serum-free medium. After 24 h, the cells were stimulated with IGF-I (10 ng/ml; Upstate Biotechnology, Inc., Lake Placid, NY) for 1 or 3 h in the presence or absence of 10 ng/ml rapamycin The cells were scraped into 1 ml of ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 50 mM beta -glycerophosphate, 1 mM EGTA, 50 mM NaF, 10 mM sodium pyrophosphate, 0.1 mM Na3VO4, 50 mM okadaic acid, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 2 mM benzamidine, and 10 µg/ml soybean trypsin inhibitor). Lysis was accomplished by three freeze-thaw cycles. To bind eIF4E, 25 µl of 7-methyl-GTP-Sepharose (Amersham Biosciences) was added to the lysates, which were then incubated overnight on a rotator at 4 °C. The complexes were pelleted by centrifugation and washed three times with lysis buffer. To elute eIF4E from the Sepharose, 50 µl of SDS-PAGE loading buffer was added to the samples, which were then heated to 95 °C for 3 min. Samples were analyzed for 4E-BP and eIF4E by SDS-PAGE and Western blot with standard chemiluminescent methods, as described above.

Assay of Ribosomal p70 S6 Kinase Activity-- Assays were performed as described previously (37). Briefly, 2 × 106 cells were seeded in 75-cm2 flasks and allowed to attach overnight. Initial experiments determined that exposure to rapamycin for 1 h resulted in maximal inhibition of p70 S6 kinase activity. Cells were exposed for 1 h to rapamycin (100 ng/ml), washed extensively, and lysed by gentle rocking at 4 °C in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1 mM NaF) containing 10 µg/ml each aprotinin, leupeptin, and pepstatin. Lysates were centrifuged at 15,000 × g for 5 min at 4 °C to remove nuclei. Twenty microliters of a mixture of anti-p70 S6 kinase polyclonal antibody (2 µg; Santa Cruz Biotechnology, Santa Cruz, CA) and protein A-conjugated Sepharose beads were added to the supernatant; the mixture was kept on a rotator overnight at 4 °C. After centrifugation, the beads were washed twice with PBS and resuspended in 20 µl of p70 S6 kinase assay buffer (20 mM MOPS, pH 7, 25 mM beta -glycerol phosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM dithiothreitol). The S6 kinase assay kit (Upstate Biotechnology) was used according to the manufacturer's instructions to assay p70 S6 kinase activity.

Induction and Assay of c-Myc-- Cells were plated in 2 ml of medium in six-well plates (Corning, NY) at a density of 5 × 105 cells/35-mm well. After overnight incubation at 37 °C and 5% CO2, medium was removed from adherent cells, and 2 ml of serum-free RPMI 1640 supplemented with 2 mM L-glutamine was added to each well. After 24 h, the cells were stimulated by adding serum to a final concentration of 10% with or without rapamycin (100 ng/ml). Cells were further incubated for the appropriate time periods, washed with ice-cold PBS, and processed as described above for Western analysis with anti-c-Myc monoclonal antibody 1-9E10 (37).

Northern Blot Analysis of 4E-BP-- Total RNA was isolated from Rh30 cells, the Rapa10K clone, and the Rapa10K/Rev clone (a revertant to rapamycin sensitivity) by using the RNeasy kit (Qiagen). Briefly, 1.0 × 107 cells were washed with PBS, treated with trypsin, and pelleted by centrifugation at 300 × g for 5 min. The pellets were lysed and processed according to the manufacturer's instructions. Ten µg of total RNA from each sample was separated by electrophoresis on a denaturing agarose gel containing 0.66 M formaldehyde. The RNA was transferred to a nylon membrane (Gene Screen Plus, PerkinElmer Life Sciences) by capillary transfer in 10× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate) buffer. The samples were hybridized with a random-primed probe (RediPrime, Amersham Biosciences) synthesized from a 375-base pair NotI-ApaI fragment containing the coding sequence for the 4E-BP1 protein (provided generously by Robert T. Abraham, Duke University, NC). A probe to the human housekeeping gene encoding glycerol-3-phosphate dehydrogenase (CLONTECH) was synthesized and served as a control for RNA loading. Hybridization was conducted in ExpressHyb buffer (CLONTECH) according to the manufacturer's instructions. The blots were washed at a final stringency of 0.5× SSC, 1% SDS at 65 °C, and autoradiograms were produced.

Assay of 4E-BP Synthesis in Rapamycin-sensitive and Rapamycin-resistant Rh30 Cells-- Rh30 and Rapa10K cells were plated in 100-mm tissue culture dishes. When cells had reached 80-90% confluence, the culture medium was aspirated. The cells were washed twice with 10 ml of prewarmed (37 °C) pulse-labeling medium (Sigma), 5 ml of prewarmed pulse-labeling medium was added, and samples were incubated for 15 min in a humidified incubator (37 °C, 5% CO2) to deplete intracellular pools of methionine. Medium was aspirated, and 2 ml of prewarmed pulse-labeling medium containing [35S]methionine (0.2 mCi/ml; PerkinElmer Life Sciences) was added. Samples were incubated (37 °C, 5% CO2) for up to 4 h, washed twice with 10 ml of cold PBS, and lysed at 4 °C for 1 h in 1 ml of cell lysis buffer (Cell Signaling, Beverly, MA) containing protease inhibitors. Lysates were transferred to 1.5-ml Eppendorf tubes, disrupted by sonication, and centrifuged at 17,500 × g for 15 min. Protein A/G (20 µl; Santa Cruz) and normal rabbit or mouse IgG (2 µg; Santa Cruz) were added to the supernatants, and samples were rotated at 4 °C for 1 h. The complexes were pelleted at 12,500 × g for 5 min. Rabbit polyclonal anti-4E-BP antibody (20 µg; Zymed Laboratories Inc., South San Francisco, CA) or mouse monoclonal anti-beta -tubulin antibody (20 µg; Sigma) were added, and samples were rotated at 4 °C overnight. Protein A/G-conjugated beads (30 µl) were added, and samples were rotated for 3 h at 4 °C. After centrifugation, the beads were washed twice with 500 µl of cold lysis buffer and twice with 500 µl of cold PBS. The pellets were resuspended in 20 µl of SDS sample buffer and separated in a 12% Bio-Rad Ready Gel. The gel was dried at 80 °C for 1 h, and the radiolabeled products were quantified by scanning of the resulting autoradiograms.

Stable Transfection of HCT8 Colon Carcinoma Cells with 4E-BP Expression Vector-- HCT-8 cells were plated at a density of 3 × 106 cells per Nunc T75 peel-top flask (Nalge Nunc International, Rochester, NY) and transfected for 18 h under serum-free conditions in Dulbecco's modified Eagle's medium with FuGENE 6 transfection reagent (Roche Molecular Biochemicals). The 4E-BP1 construct of interest, pcDNA3-PHAS-I (a gift from Robert T. Abraham), was transfected at a concentration of 10 µg/75-cm2 flask. Cells were then washed with Hanks' solution and fed with RPMI 1640 containing 10% dialyzed FBS and 2 mM L-glutamine. After 48 h, fresh medium containing 500 µg/ml G418 was added. Surviving colonies were allowed to grow for 2 weeks and were then ring-cloned and placed in 12.5-cm2 flasks in 2 ml of medium containing 10% dialyzed FBS, 2 mM L-glutamine, and 500 µg/ml G418. Ten colonies were selected for further evaluation.

Western Analysis of 4E-BP1-- Cultured cells at a logarithmic phase of growth were briefly washed with cold PBS, and samples containing 1.5 × 106 cells were lysed on ice in 75 µl of lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerophosphate, 1 mM Na3VO4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 1 tablet of protease inhibitor mixture (Roche CompleteTM Mini). Cells were frozen in liquid N2 for 5 min and thawed at 37 °C for 5 min three times and centrifuged at 17,500 rpm × g for 10 min at 4 °C; supernatants were then transferred to a fresh tube, and 25 µl of NuPAGE 4× lithium dodecyl sulfate sample buffer (Invitrogen) was added. Samples were heated for 10 min at 70 °C before centrifugation at 17,500 rpm × g for 10 min at 4 °C. Proteins were separated on a 12% bis-Tris-polyacrylamide gel (Invitrogen) and transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were incubated first with rabbit polyclonal anti-4E-BP1, (Zymed Laboratories Inc.) and then with goat anti-rabbit IgG conjugated to horseradish peroxidase (Pierce). Immunoreactive bands were visualized by using Pierce SuperSignal® chemiluminescence substrate (Pierce) and Kodak BiomaxTM MR film (Eastman Kodak Co.).

Clonogenic Assays-- Cultured cells were plated in triplicate (3000 cells/35-mm dish) in 6-well Falcon culture plates (BD PharMingen). After 24 h, the medium was aspirated from the adherent cells, and 2 ml of RPMI 1640 containing 10% FBS, 2 mM L-glutamine, and rapamycin (serial dilutions, 0-10,000 ng/ml) was added to each well. After 7 days, cells were washed with 0.9% saline solution and stained with 0.1% crystal violet. Colonies were enumerated by using an AlphaImager 2000TM (Alpha Innotech Corp.).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Selection of Rapamycin-resistant Clones-- The alveolar rhabdomyosarcoma cell line Rh30 is exquisitely sensitive to rapamycin (4, 33) and has been used as a model system for studying acquired rapamycin-resistant phenotypes. Cells were grown in increasing concentrations of rapamycin to obtain lines that proliferated in the presence of 5000 or 10,000 ng/ml rapamycin (IC50 ~ 10,000- to 20,000-fold greater than that of the parental lines). Three clones were further characterized; they were Rapa10K and C2, which were selected without mutagenesis, and C4, which was selected after treatment with ethane methylsulfonate.

Rapamycin Resistance Is Unstable-- Rapa10K, C2, and C4 cells growing in rapamycin (10,000 ng/ml) were washed extensively in Hepes-buffered saline solution, plated, and allowed to attach to the surface of the flask overnight in drug-free medium. The cells were then cultured for 7 additional days in the presence of drug before they were stained and enumerated or were grown without rapamycin for 6 or 10 weeks and were then retested for sensitivity. Fig. 1A shows the rapid decrease in IC50 in the absence of rapamycin, signifying reversion to sensitivity. Within 6 weeks of drug withdrawal, the rapamycin IC50 of Rapa10K cells decreased from ~1400 ng/ml to <1 ng/ml. Similar results were obtained with C2 and C4 clones. Complete concentration-response curves for Rapa10K and C2 cells are shown in Fig. 1, B and C. These data demonstrate that cells selected for acquired resistance to rapamycin with or without prior mutagenesis revert to a sensitive phenotype rapidly in the absence of drug. Clones that reverted to rapamycin sensitivity were designated Rapa10K/Rev, C2-Rev, and C4-Rev.


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  		Fig. 1.   Rapamycin resistance is unstable in the absence of selection pressure. A, rapamycin-resistant clones Rapa10K (), C2 (black-square), and C4 (black-down-triangle ) were selected for ability to proliferate in 10,000 ng/ml rapamycin. Drug was removed, cells were grown overnight, and sensitivity to rapamycin was determined at that time (week 0) and after an additional 6 or 10 weeks of culture. Data points show the mean concentration of rapamycin that inhibited proliferation by 50% (IC50) in triplicate experiments. B, Rapa10K cells were cultured in media containing 10,000 ng/ml rapamycin, and their sensitivity to increasing concentra- tions of rapamycin was determined after culture without drug for 16 h (black-triangle) or 6 weeks (). Sensitivity of parental Rh30 cells is shown for comparison (black-square). The IC50 values were 1372 ng/ml (Rapa10K after 16 h), 0.95 ng/ml (Rapa10K after 6 weeks), and 0.69 ng/ml (Rh30). Each point represents the mean ± S.D. of three determinations. C, C2 cells were cultured in media containing 10,000 ng/ml rapamycin. Their sensitivity to increasing concentrations of rapamycin was determined after culture without the drug for 16 h (black-triangle), 6 weeks (), or 10 weeks (black-square). The IC50 values were 9933 ng/ml at 16 h, 121 ng/ml at 6 weeks, and 2.9 ng/ml at 10 weeks. Each point represents the mean ± S.D. of three determinations.

Rapamycin-resistant Cells Down-regulate 4E-BP Expression-- We next sought to characterize the mechanism of resistance. We reported previously that intrinsic resistance was not a consequence of reduced drug accumulation or retention or of altered levels of mTOR (37). However, the correspondence between acquired resistance and elevated intracellular c-Myc in Rapa10K (37) suggested that regulation of the eIF4E pathway was altered. In cells starved of growth factor, 4E-BPs rapidly become hypophosphorylated and associate with eIF4E, thereby preventing binding of eIF4E to the scaffold protein eIF4G and inhibiting initiation of eIF4E-dependent translation. Phosphorylation of 4E-BPs by growth factors is mTOR-dependent; hence, rapamycin inhibits growth factor-induced dissociation of 4E-BPs and eIF4E. To examine the interaction of 4E-BP with eIF4E in Rh30 cells, we starved the Rh30 clones Rapa10K and Rapa10K/Rev of serum for 24 h and then stimulated them with IGF-I in the presence or absence of rapamycin. Lysates were incubated with 7-methyl-GTP-Sepharose beads to bind eIF4E and any associated 4E-BP. The results, shown in Fig. 2, demonstrate that 4E-BP1 and eIF4E are associated in unstimulated Rh30 cells but are rapidly dissociated by IGF-I stimulation. The IGF-I-mediated dissociation was completely blocked by exposure to rapamycin before stimulation. The results with Rapa10K were striking. Although rapamycin treatment would be expected to increase the association of 4E-BP1 with eIF4E, this association was dramatically suppressed in Rapa10K, regardless of the experimental condition. However, the quantities of eIF4E and beta -tubulin in the resistant Rapa10K cells were similar to those in the parental Rh30 cells. Importantly, association of 4E-BP1 with eIF4E in the revertant Rapa10K/Rev cells was similar to that observed in the parental Rh30 line. Similar results were obtained in C2 and C4 clones and their revertants (data not shown).


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  		Fig. 2.   Binding of 4E-BP1 to eIF4E is reduced in rapamycin-resistant cells. Rh30, Rapa10K, and Rapa10K/Rev cells were starved of serum overnight and were then stimulated for 1 or 3 h with IGF-I (10 ng/ml) with or without prior incubation with rapamycin (Rap; 1 h; 100 ng/ml). After eIF4E was isolated from lysates by using 7-methyl-GTP-Sepharose beads, 4E-BP and eIF4E were assayed by Western blot analysis with beta -tubulin as a loading control. Results shown are representative of three experiments.

To determine whether the dramatic decrease in 4E-BP binding to eIF4E in rapamycin-resistant clones was caused by suppression of 4E-BP1 expression, we assayed 4E-BP1 in total cell extracts. To obtain results that were comparable with those obtained in the previous experiment, we used parallel conditions of serum starvation and IGF-I stimulation with or without rapamycin. The results of Western blot analyses demonstrated that resistance was associated with substantially decreased levels of 4E-BP1 (Fig. 3A). In Rapa10K cells, the quantity of 4E-BP1 was markedly less than that of eIF4E or beta -tubulin and was barely detectable. Rapa10K/Rev cells contained more 4E-BP1 than did Rapa10K but less than did parental cells. Essentially identical results were obtained by using a polyclonal antibody to the highly conserved carboxyl terminus of 4E-BP that recognizes all 4E-BP isoforms (data not shown). Hence, results obtained with isoform-specific antibody apply to all 4E-BPs.


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  		Fig. 3.   Rapamycin resistance corresponds to decreased levels of 4E-BP. A, Rh30, Rapa10K, and Rapa10K/Rev cells were serum-starved overnight and then stimulated for 1 or 3 h with IGF-I (10 ng/ml) with or without prior incubation with rapamycin (Rap; 1 h; 100 ng/ml). Total cell lysates were analyzed for 4E-BP, eIF4E, and beta -tubulin (loading control). B, Northern blot analysis of total RNA from Rh30, Rapa10K, and Rapa10K/Rev cells with probes for 4E-BP and G3PDH RNA. C, levels of 4E-BP protein in Rh30, C2, and C2-Rev clones determined by Western blot analysis as described in panel A.

Northern blot analysis showed no decrease in steady-state levels of 4E-BP transcript in Rh30, Rapa10K, and Rapa10K/Rev cells (Fig. 3B). Thus, 4E-BP protein levels in resistant clones appear to be suppressed through a posttranscriptional mechanism. Rapamycin-resistant C2 cells also showed reduced levels of 4E-BP protein, whereas rapamycin-sensitive C2-Rev contained levels of 4E-BPs similar to those in Rh30 cells (Fig. 3C). Similar results were obtained with C4 and C4-Rev clones (not shown). Collectively, these results suggest that acquired resistance to rapamycin is a result of down-regulation of 4E-BP protein expression. An increase in 4E-BP expression when the selective pressure is removed coincides with the return of sensitivity.

The Rate of 4E-BP Synthesis Is Reduced in Resistant Cells-- We next investigated whether steady-state levels of 4E-BP were reduced in resistant clones because of decreased synthesis or because of increased degradation. Rh30 and Rapa10K cells were incubated with [35S]methionine for up to 4 h in methionine-free medium. Total beta -tubulin was assayed in cell lysates by Western blot analysis, 4E-BP and beta -tubulin were immunoprecipitated, and incorporated radioactivity was quantified by autoradiography. Representative results are shown in Fig. 4. The rapamycin-resistant Rapa10K cells incorporated less radiolabel into 4E-BP than did parental Rh30 cells, although incorporation of radiolabel into tubulin was slightly enhanced. The beta -tubulin content of parental and rapamycin-resistant clones, as determined by Western blot analysis, was similar. The rate of 4E-BP synthesis in Rapa10K cells was about 50% that in parental cells after results were normalized by tubulin values. In other studies, we found that PS341, an inhibitor of the 20S proteasome (40), did not alter 4E-BP levels in Rapa10K cells (data not shown).


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  		Fig. 4.   Low 4E-BP levels reflect a reduced rate of synthesis. A, autoradiograms of immunoprecipitated 35S-4E-BP and 35S-beta -tubulin (upper and center panels) and total cellular beta -tubulin (Western blot) from Rh30 and Rapa10K cells. B, incorporation of [35S]methionine into 4E-BP in Rh30 and Rapa10K cells. Results were normalized to beta -tubulin values. Incorporation of [35S]methionine into beta -tubulin in Rh30 and Rapa10K cells. Results were normalized to beta -tubulin values.

Resistance Is Not Associated with Consistent Changes in the p70 S6 Kinase Pathway-- To determine whether the ribosomal p70 S6 kinase pathway downstream of mTOR is altered in rapamycin-resistant cells, we used two assays. The quantity and activity of p70 S6 kinase were determined in parental rapamycin-resistant clones and in their revertant clones. In resistant clones, quantities of p70 S6 kinase were similar to (Rapa10K) or less (C2 and C4) than those in wild-type Rh30 cells after adjustment for protein loading (Fig. 5A shows the results of a representative experiment). The histogram in Fig. 5B shows the combined results of three separate experiments. We next assessed the stimulation of p70 S6 kinase activity by IGF-I in wild-type drug-resistant clones maintained in rapamycin and in revertant clones (Fig. 5C). Cells were washed, serum-starved overnight, and stimulated with IGF-I in the presence or absence of rapamycin. In Rh30 cells, IGF-I stimulated p70 S6 kinase activity, and this activity was suppressed by rapamycin. In the C2 and C4 clones, stimulation with IGF-I did not increase p70 activity. This result is consistent with prolonged rapamycin retention and delayed recovery of mTOR activity (37). In contrast, IGF-I stimulated p70 S6 kinase activity in both revertant clones, and this stimulation was blocked by rapamycin. These results indicate that induction of p70 S6 kinase activity by IGF-I is completely suppressed in cells maintained in high concentrations of rapamycin. Furthermore, there was no detectable increase in p70 S6 kinase protein or activity that would be expected to accompany rapamycin resistance.


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  		Fig. 5.   Rapamycin resistance is not associated with consistent changes in ribosomal p70 S6 kinase levels or activity. A, Western blot analysis of p70 S6 kinase levels in parental, resistant, and revertant clones. After the membranes had been stripped of bound anti-p70 S6 kinase antibody, they were incubated with an anti-beta -tubulin antibody. The quantity of beta -tubulin was used to control for differences in protein loading. Results shown are those of a representative experiment. B, integration of data from three independent Western blot analyses of total cellular p70 S6 kinase. Values are expressed relative to those of beta -tubulin. C, assays of p70 S6 kinase activity. Rh30, C2, and C4 clones and their revertant clones were serum-starved overnight, incubated for 1 h with or without 100 ng/ml rapamycin (Rap), and stimulated for 30 min with IGF-I. Results are the means ± S.D. of three experiments. DPM, disintegrations/min.

Rapamycin Resistance Is Associated with Increased c-Myc Expression-- We had noted previously that c-Myc levels were elevated in Rapa10K. As shown in Fig. 6, the quantity of c-Myc in Rh30 cells was increased by serum stimulation, and this effect was inhibited by rapamycin. In Rapa10K cells, the quantity of c-Myc was ~4-fold that in Rh30 cells, and this increase was less inhibited by rapamycin. Interestingly, c-Myc levels appeared to be slightly higher in Rapa10K/Rev cells than in Rapa10K cells. Similar results were obtained with the C2 clone; c-Myc expression was greater in C2 than in Rh30 and was not decreased by rapamycin treatment. Levels of c-Myc in C2-Rev were also high. Notably, overnight serum starvation appeared not to reduce the level of c-Myc protein substantially in C2 or the revertant lines. Consequently, serum stimulation with or without rapamycin treatment had little effect on c-Myc levels in C2 and C2-Rev clones. Thus, elevated c-Myc expression is associated with rapamycin resistance but is not necessarily causal. The finding that c-Myc expression is elevated in the revertants, which are sensitive to growth inhibition by rapamycin, suggests that inhibition of c-Myc translation is not the key mechanism of arrest of cells in G1 phase.


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  		Fig. 6.   Acquired resistance is associated with increased expression of c-Myc. Cells were serum-starved overnight and stimulated for 1 to 7 h with IGF-I (10 ng/ml) with or without prior incubation with rapamycin (Rap; 1 h; 100 ng/ml). Western blots of lysates were probed with antibodies to for c-Myc and beta -tubulin (loading control). A, Rh30, Rapa10K, and Rapa10K/Rev cells. B, C2 and C2-Rev cells.

Malignancy-associated Characteristics Are Increased in Rapamycin-resistant Cells-- Rapamycin-resistant and revertant clones exhibited enhanced anchorage-independent growth in soft agar, which is consistent with dysregulation of c-Myc. Table I shows the growth characteristics (colony count and colony size) of Rapa10K and C2 and their respective revertants. The clonogenicity of Rapa10K and its revertant was 5-7-fold that of the parental Rh30 cells. The clonogenicity of C2 was 13-fold, and the clonogenicity of C2-Rev was 36-fold that of Rh30 cells. Notably, colony size (area) was consistent among parental, resistant, and revertant clones; this finding suggests that increased frequency of colony formation is not a consequence of enhanced growth rate. These results are consistent with previous reports that overexpression of eIF4E causes malignant transformation (41-43).

                              
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Table I
Anchorage-independent growth of Rh30 clones selected for rapamycin resistance and of clones that reverted to rapamycin sensitivity

Overexpression of 4E-BP Reverses Intrinsic Resistance to Rapamycin-- The findings above implicate a low 4E-BP·eIF4E ratio in resistance to rapamycin. If so, then overexpression of 4E-BP could potentially restore rapamycin sensitivity to Rapa10K cells. However, the selection of stable clones that overexpress 4E-BP would necessitate growth in rapamycin-free medium, which in turn would rapidly induce reversion to rapamycin sensitivity (Fig. 1). As an alternative approach, we examined the 4E-BP·eIF4E ratio in a series of colon adenocarcinoma cells shown to be highly resistant to rapamycin (4). Fig. 7A shows quantities of 4E-BP and eIF4E relative to tubulin in six colon carcinoma lines intrinsically resistant to rapamycin (IC50 1280 to >10,000 ng/ml) and three tumor cell lines sensitive to rapamycin (Rh18, Rh30, SJ-G2; IC50 0.1-0.5 ng/ml). Clearly, in four of the colon carcinoma lines, the ratio of 4E-BP·eIF4E is low, consistent with resistance, whereas in the GC3 and VRC5 colon carcinoma cells, the ratio is similar to that in sensitive cells. Hence, a low 4E-BP·eIF4E ratio cannot account for intrinsic resistance to rapamycin in either GC3 or VRC5 cell lines. To determine whether an increase in the amount of 4E-BP could sensitize cells to rapamycin, we transfected HCT8 cells with a 4E-BP expression plasmid and selected stable clones by growth in medium containing G418. As shown in Fig. 7B, several clones (2, 4, and 5) were identified that had increased expression of 4E-BP, whereas two G418-resistant clones (1 and 3) had 4E-BP levels similar to that of parental HCT-8 cells. Levels of eIF4E were similar in all clones identified. The rapamycin sensitivity of each clone is shown in Fig. 6C. Cells were grown in increasing concentrations of rapamycin (0-10,000 ng/ml), and colonies were counted after 7 days. HCT8 was highly resistant to rapamycin, as expected, as were clones C1 and C3, which did not overexpress 4E-BP (IC50 > 10,000 ng/ml). In contrast, clones C2, C5, and C4 were sensitive to rapamycin, having IC50 values of 5, 8, and 30 ng/ml, respectively. The rapamycin sensitivity corresponded to 4E-BP·eIF4E ratios in these clones.


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  		Fig. 7.   Overexpression of 4E-BP abrogates resistance to rapamycin. A, Western blot analysis of 4E-BP, eIF4E, and tubulin (loading control) in cell lines that have different intrinsic sensitivities to rapamycin. Colon carcinoma cell lines CaCo2, GC3/c1, HCT8, HCT29, HCT116, and VRC5/c1 are intrinsically resistant to rapamycin (4), with IC50 concentrations > 1200 ng/ml. Pediatric solid tumor lines SJ-G2 (glioblastoma) and Rh18 and Rh30 (rhabdomyosarcoma) are sensitive to rapamycin (IC50 < 1 ng/ml). B, expression of 4E-BP and eIF4E in HCT8 clones stably transfected with a 4E-BP expression plasmid (pcDNA3-PHAS-I). Expression of 4E-BP was greater in clones C2, C4, and C5 than in parental HCT8 cells, but expression was similar in parental and C1 and C3 transfected clones. C, sensitivity to rapamycin. Cells were plated at low density in increasing concentrations of rapamycin, and colonies were counted after 7 days of exposure to rapamycin. , parental HCT8; open circle , clones C1; black-square, C2; , C3; black-triangle, C4; triangle ; C5. Each point is the mean ± S.D. of three determinations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The work reported here provides the first demonstration that acquired resistance and, in some cases, intrinsic resistance to rapamycin in malignant cells is related to a low cellular ratio of 4E-BP to eIF4E. Furthermore, we have shown that in Rapa10K cells, the decreased quantity of 4E-BP is caused in part by a reduced rate of 4E-BP synthesis.

Because rapamycin potently inhibits T-cell activation, it has been extensively used as an immunosuppressant (1-3). More recently, rapamycin and its derivatives have drawn interest as potential cancer chemotherapy agents. The rapamycin ester CCI-779 is currently in Phase II clinical trials. Mutagenesis selection studies in yeast have identified dominant mutations that reduce rapamycin binding to its immunophilin or to TOR. Dominant mutations in mammalian cells are consistent with mutations in mTOR that reduce rapamycin-FKBP binding. However, the mechanism(s) by which cells acquire rapamycin resistance in the absence of mutagens has been reported only for murine BC3H1 cells. In those cells, resistance was associated with decreased levels of the cyclin-dependent kinase inhibitor p27Kip1 (34). Consistent with this finding, p27-/- murine embryo fibroblasts were also somewhat resistant to rapamycin (34).

We demonstrated previously that intrinsic resistance to rapamycin is not associated with elevated mTOR levels or decreased drug accumulation (37). In Rh30 cells, sensitivity to rapamycin was associated with rapamycin-induced inhibition of c-Myc translation (37), and acquired resistance was associated with increased c-Myc expression. The studies described here sought to further characterize the mechanism of resistance and to identify which of the two pathways downstream of mTOR must be impeded to inhibit cell growth. Rh30 cells are exquisitely sensitive to rapamycin, with an IC50 value of ~0.5 ng/ml (4). Furthermore, the marked rapamycin resistance of Rh30 cells expressing a rapamycin-resistant mTOR (S2035I) suggests that at submicromolar concentrations, rapamycin acts specifically through inhibition of mTOR (33). We characterized two clones, Rapa10K and C2, that were able to proliferate at drug concentrations of 10,000 ng/ml and an additional clone, C4, that was selected for rapamycin resistance after mutagenesis with ethane methylsulfonate. When grown in drug-free medium for 6 or 10 weeks, these resistant clones became sensitive to rapamycin; therefore, resistance acquired with or without ethane methylsulfonate treatment was unstable. Our preliminary characterization had shown increased levels of c-Myc in Rapa10K cells (37). Because translation of c-Myc is dependent on eIF4E (43), we began by investigating possible changes in regulation of the eIF4E pathway. Rapamycin prevents IGF-I-induced phosphorylation of 4E-BP and the dissociation of 4E-BP from eIF4E (10, 39). In Rh30 cells, we found that IGF-I stimulated the release of 4E-BP from eIF4E and that this effect was prevented by rapamycin. In serum-starved Rapa10K cells, only a barely detectable quantity of 4E-BP was bound to eIF4E, and treatment with IGF-I did not alter this. The association of 4E-BP with eIF4E increased toward the level observed in parental Rh30 cells as cells reverted to rapamycin sensitivity. Similar results were obtained with C2 and C2-Rev cells.

Levels of 4E-BP were decreased in rapamycin-resistant cells. Both Rapa10K and C2 clones contained markedly less 4E-BP than did Rh30 cells. In revertant clones, 4E-BP increased to levels near (Rapa10K) or equal (C2-Rev) to those of parental cells. Similar results were obtained with C4 and its revertant. Parallel assays of 4E-BP that used polyclonal antibodies that recognize different 4E-BP epitopes yielded similar results; therefore, it is unlikely that mutations or posttranslational modifications in 4E-BP altered its affinity for these reagents. Northern blot analysis showed similar steady-state levels of 4E-BP transcript in Rh30, Rapa10K, and Rapa10K/Rev cells; therefore, 4E-BP1 levels were not reduced because of transcriptional repression. The incorporation of [35S]methionine into immunoprecipitated 4E-BPs was markedly less in Rapa10K cells than in parental Rh30 cells; this finding was consistent with reduced 4E-BP levels in resistant cells. In contrast, the rate of beta -tubulin synthesis was slightly greater in Rapa10K than in parental cells; hence, rapamycin resistance was not associated with a general decrease in protein synthesis. These results suggest that synthesis of 4E-BPs is decreased in Rapa10K cells.

Unlike the eIF4E pathway, the ribosomal p70 S6 kinase pathway showed no consistent alteration. Cells with acquired resistance to rapamycin do not show overexpression or constitutive activation of ribosomal p70 S6 kinase.

Rapa10K and C2 clones had increased c-Myc protein; this finding was consistent with a decrease in 4E-BP and potential dysregulation of eIF4E expression (44). Interestingly, c-Myc levels did not decrease when cells reverted to rapamycin sensitivity. This finding suggests that rapamycin does not inhibit the growth of Rh30 cells by reducing c-Myc translation These results could suggest that in revertant cells an internal ribosome entry sequence in c-myc transcripts that functions normally during mitosis (48) could be used (45-47). The mechanism responsible for dysregulation of c-Myc expression in these revertant clones remains to be determined.

Overexpression of eIF4E transforms NIH3T3 fibroblasts and rat embryo fibroblasts and cooperates with v-myc or E1A in the transformation of primary rat fibroblasts (41.42). Ras also mediates eIF4E-induced transformation (49), and increased eIF4E expression has been proposed as a contributor to malignant progression (50). Rh30 cells express the Pax3-FKHR chimeric transcription factor, which is thought to play an initial role in transforming these cells (51, 52). It was of interest, therefore, to determine whether the decreased regulation of eIF4E expression that is associated with rapamycin resistance enhances the anchorage-independent growth of cells. Our results showed an increase in the frequency of colony formation but not in the rate of colony growth in both rapamycin-resistant and revertant cells. Maintenance of this malignant phenotype in cells that revert to rapamycin sensitivity appears to correspond to continued dysregulation of c-Myc expression. These results suggest also that the eIF4E pathway downstream of mTOR may be important in establishing the increased colony-forming ability characteristic of an increased malignant phenotype. Conversely, inhibition of mTOR may suppress this characteristic.

We next investigated whether increased 4E-BP levels could confer rapamycin sensitivity. We did not overexpress 4E-BP in Rapa10K cells, because rapamycin-free selection conditions would cause spontaneous reversion to rapamycin sensitivity. Instead, we sought a cell line with constitutive low expression of 4E-BP. We had previously identified colon carcinoma cell lines that have high intrinsic resistance to rapamycin (4). Of the six tumor lines examined, four had very low levels of 4E-BP. In contrast, levels of eIF4E in sensitive tumor cell lines were similar to those in resistant tumor cell lines. These findings suggested that low 4E-BP·eIF4E ratios might represent one mechanism of intrinsic resistance in colon cancer cell lines. To test this hypothesis directly, we selected HCT8 clones that stably expressed 4E-BP. Three clones overexpressed 4E-BP, whereas two had 4E-BP levels similar to those of parental HCT8 cells. The growth rate and colony formation characteristics of all the clones were similar. However, the clones that overexpressed 4E-BP were markedly more sensitive to rapamycin. The C2 and C5 clones, which had the highest levels of 4E-BP expression, had IC50 values of only ~5 ng/ml rapamycin. In contrast, the IC50 values of clones that did not overexpress 4E-BP were >10,000 ng/ml. These results support the premise that a decreased 4E-BP·eIF4E ratio may be a determinant of intrinsic or acquired resistance. Furthermore, it is the eIF4E pathway rather than the p70 S6 kinase pathway that is crucial to rapamycin-induced growth inhibition, at least in Rh30 and HCT8 cells.

    ACKNOWLEDGEMENTS

We thank Nahum Sonenberg and Robert Abraham for generously providing reagents and advice. We also thank Sharon Naron for editorial assistance in preparing this manuscript.

    FOOTNOTES

* This work was supported in part by United States Public Health Service Grants CA23099, CA77776, and CA21765 (Cancer Center Support Grant) from the NCI, National Institutes of Health and by the American Lebanese Syrian Associated Charities (ALSAC).

Dagger To whom correspondence should be addressed: Dept. of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105-2794. Tel.: 901-495-3440; Fax: 901-521-1668; E-mail: peter.houghton@stjude.org.

Published, JBC Papers in Press, February 14, 2002, DOI 10.1074/jbc.M110782200

    ABBREVIATIONS

The abbreviations used are: TOR, target of rapamycin; mTOR, the mammalian target of TOR; EIF, eukaryotic initiation factor; 4F-BP, 4F-binding protein; IGF-I, insulin-like growth factor I; FBS, fetal bovine serum; Rapa10K, rapamycin, 10,000 ng/ml; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; Rev, revertant; IC50, 50% inhibitory concentration; FKBB-12, FK506 binding protein (12 kDa).

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RESULTS
DISCUSSION
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