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Experimental Therapeutics |
Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105-2794
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and 2 ß-subunits. The tyrosine kinase catalytic site and the ATP-binding site are located in the cytoplasmic portion of the ß-subunit. The tyrosine kinase activity of the ß-subunit is stimulated when IGF-I binds to the -subunit. Intracellular substrates of the IGF-IR include the insulin receptor substrates IRS-1 and IRS-2, and the SH2-containing protein Shc. Phosphorylation of these proteins results in their interaction with other signaling molecules, such as the adapter Grb2, that are, in turn, coupled to effector molecules, such as the guanine nucleotide exchange factor Sos (4, 5, 6)
and PI3K (7
, 8)
. The activated Shc-Grb2-Sos complex activates the small GTP-binding protein Ras, and activated Ras initiates a cascade of Ser/Thr kinases, some of which eventually translocate to the nucleus to stimulate gene expression (9
, 10)
. The extracellular signal-regulated MAPKs, Erk1 and Erk2, are key intermediates in the propagation of signals from many growth factor receptors to the nucleus (9
, 11)
. Erk proteins are activated by MEK, a dual function kinase that phosphorylates tyrosine and threonine residues of Erk (12
, 13)
downstream of Ras and Raf1. IRS-1 and IRS-2 lie upstream of a signaling cascade involving PI3K. Phosphoinositides phosphorylated by PI3K recruit PDKs such as PDK1 to the plasma membrane. Evidence suggests that phosphorylation of Thr308 and Ser473 of Akt by PDK1 and PDK2 activates Akt (14, 15, 16) . This event results in Akt phosphorylation of its downstream substrates such as GSK-3 (17) , 6-phosphofructo-2-kinase (18) , the Bcl2 family member Bad (19) , caspase-9 (20) , nitric oxide synthase (21 , 22) , and the winged-helix family of FKHRL1 transcription factors (23) . Activation of these substrates leads to glucose transport, glycolysis, glycogen synthesis, and cell survival (24 , 25) . Thus, IGF-IR, when activated by its ligands, plays an important role in the growth of cells by inducing mitogenesis and transformation, and by protecting cells from various apoptotic injuries (26) . Several reports have documented recently the involvement of IGF-I in regulating apoptosis (27) induced by various stimuli, including physiological stress (28) , hyperosmosis (29 , 30) , chemotherapy (31) , and DNA damage caused by chemotherapeutic drugs or UV-B radiation (32, 33, 34) .
Additional evidence of the role of IGF-I in regulating apoptosis has been provided by studies involving rapamycin, an immunosuppressive macrocyclic lactone that specifically inhibits the activity of mTOR, a Ser/Thr kinase downstream of PI3K. Inhibition of mTOR leads to G1 arrest of many malignant cell lines, and currently analogs of rapamycin are being investigated as cancer therapeutic agents. We have reported previously that rapamycin selectively induces apoptosis of tumor cells that express mutant p53 and are grown under serum-free conditions (35) . However, the addition of IGF-I to the growth medium completely protects Rh1 cells from rapamycin-induced apoptosis. Therefore, we are interested in understanding how IGF-I protects Rh1 cells from apoptosis induced by rapamycin.
Receptor tyrosine kinases such as those for the receptors of IGF-I, insulin, and PDGF stimulate nuclear events by activating cascades of protein kinases (4) . EGF activates the PI3K-Akt signaling pathway in several EGF receptor-overexpressing cells such as prostate cancer cells (36) , epidermoid cancer cells (37) , and ovarian cancer cells (38) . Extensive studies have shown that the pathways used for all of the functions of IGF-IR appear to overlap considerably. Because the Ras-Erk1-Erk2 and PI3K-Akt pathways are the two major implicated in survival signaling in a wide variety of cancer cells (reviewed in Ref. 2 ), we sought to determine whether one or both of these pathways is required for the IGF-I-mediated prevention of rapamycin-induced apoptosis of Rh1 and Rh30 cells.
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MATERIALS AND METHODS |
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Cell Lines and Growth Conditions.
The human rhabdomyosarcoma cell lines Rh1 and Rh30 have been described (35)
. Briefly, Rh1 and Rh30 cells were grown in antibiotic-free RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and 2 mM L-glutamine (BioWhittaker) at 37°C in an atmosphere of 5% CO2. For experiments in which cells were deprived of serum overnight, cell monolayers were washed with RPMI 1640 containing 2 mM L-glutamine and incubated in the same medium. For prolonged serum-free experiments, cells were cultured in modified N2E (MN2E) medium (DMEM/F-12; 1:1 mixture) supplemented with 1 µg/ml human holo transferrin, 30 nM sodium selenite, 20 nM progesterone, 100 µM putrescine, 30 nM vitamin E phosphate, 50 µM ethanolamine, and 1 mg/ml BSA (Sigma, St. Louis, MO). Cells in MN2E medium containing 5 µg/ml bovine fibronectin (Sigma) were plated and allowed to attach overnight at 37°C in a humidified 5% CO2 atmosphere.
ApoAlert Assay.
We used the ApoAlert Annexin V-FITC Apoptosis kit (Clontech) to evaluate the extent of apoptosis within cell populations. Rh1 cells alone, Rh1 cells transduced with MSCV-I-GFP or MSCV-I-GFP/RasN17, or Rh1 cells transfected with pUSE or pUSE-dnAkt (1.5 x 106/162-cm2 flask) were grown overnight in MN2E medium. On day 1, cells were treated with DMSO (0.1%; vehicle control) or rapamycin (100 ng/ml). The appropriate growth factors recombinant human IGF-I and PDGF (both from Upstate Biotechnology) and the sodium salt of human recombinant insulin and recombinant human EGF (both from Sigma) were added individually to some of the cells treated with DMSO and to some treated with rapamycin. After 4 days, the cells were trypsinized, washed with PBS, and resuspended in 200 µl of binding buffer. Cells were incubated with 10 µl of annexin V-FITC (final concentration, 1 µg/ml) and 500 ng of propidium iodide in a final volume of 410 µl. Cells were incubated at room temperature in the dark for 10 min before flow cytometric analysis (FACScalibur; Becton Dickinson) was performed as described (35)
. Statistical significance of differences between viable cells in control and treatment groups was tested using the Student unpaired t test.
Western Blot Analysis.
Cultured cells were briefly washed with ice-cold PBS. For the analysis of Akt, Erk1, and Erk2, cells were placed on ice and lysed in RIPA buffer [150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, and 50 mM Tris-HCl (pH 7.2)] containing 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin. For the detection of prelamin A, the cells were lysed in a urea-based buffer [6 M urea, 2% SDS, 62.5 mM Tris (pH 6.8) and 1 mM EDTA]. For the detection of Ras, cells were lysed in magnesium lysis buffer [25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 µg/ml aprotinin, 1 µg/ml leupeptin, one Complete mini protease inhibitor tablet (Boehringer Mannheim, Mannheim, Germany), 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1 mM NaF]. Cellular debris was pelleted by centrifugation at 17,500 x g for 10 min at 4°C. Protein concentration of the supernatants was measured by the bicinchoninic acid assay using BSA as the standard (Pierce, Rockford, IL).
For the analysis of Akt, p70S6K, Erk1, Erk2, and Ras, equivalent amounts of proteins from various samples were subjected to electrophoresis through a 12% SDS-polyacrylamide gel (Bio-Rad) and subsequently transferred to PVDF membranes (Immobilon; Millipore, Bedford, MA). After a 1-h incubation in 1x Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat dry milk at room temperature, the wet PVDF membranes were then incubated with the appropriate antibody: rabbit polyclonal antiserum specific for the phosphorylated Thr202 of Erk1 and the phosphorylated Tyr204 of Erk2 (dilution, 1:4,000; Cell Signaling Technology, Beverly, MA); rabbit polyclonal antiserum specific for the phosphorylated Ser473 of Akt (dilution 1:4,000; Cell Signaling Technology); rabbit polyclonal antiserum specific for the phosphorylated Thr389 of p70S6K (dilution 1:4,000; Cell Signaling Technology); and mouse monoclonal antibody (IgG2aK) to Ras or Ras10 (dilution, 1:1,000; Upstate Biotechnology, Lake Placid, NY). The secondary antibody was either the horseradish peroxidase-conjugated goat antirabbit IgG antibody (dilution, 1:10,000) or horseradish peroxidase-conjugated goat antimouse IgG antibody (dilution, 1:20,000). The Renaissance chemiluminescence agent was used to visualize bound antibody.
To ensure that equivalent amounts of protein were loaded on each gel, all of the immunoblots were treated with stripping buffer [62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM ß-mercaptoethanol] for 30 min at 50°C and then incubated with one of the appropriate antibodies: rabbit polyclonal antibody to Erk2 (K-23; dilution, 1:4000; Upstate Biotechnology), rabbit polyclonal antibody to Akt (dilution, 1:4000; Cell Signaling Technology), or mouse monoclonal antibody to ß-tubulin (dilution, 1:2000; Sigma). The secondary antibodies and detection of bound antibody were as described in the preceding paragraph.
RasN17 Retrovirus.
The DNA sequence coding for the dominant-negative form of Ras, RasN17 (39)
, was subcloned into the EcoRI site of the retroviral vector MSCV-I-GFP (40)
, which contains an internal ribosomal entry site for translation of the single transcript encoding both GFP and RasN17. Viral particles pseudotyped with the feline endogenous virus (RD114) envelope protein were then generated by transiently transfected 293T cells (41)
. Briefly, 15 µg of the helper vector PAM3E-, 12.5 µg of MSCV-I-GFP-RasN17, and 3 µg of the helper vector RD114 were transfected into 3 x 106 293T cells on a 10-cm plate by using the N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid/calcium method. After 12 h, the medium was replaced with fresh DMEM supplemented with 10% FBS. After an additional 4 h, the medium was replaced with 3 ml of complete DMEM. Thereafter, at the end of every 6 h during a 48-h period, the medium was collected and placed on ice. The collected samples were pooled, filtered, and dispensed into aliquots that were stored at -80°C. On the basis of the fluorescence of GFP in infected Rh1 cells, the titer of the virus was determined to be 5 x 105 infectious particles per ml.
Rh1 cells were transduced with 3 ml of virus in the presence of 10 µg/ml of hexadimethrine bromide (Sigma). After 3 h, the viral supernatant was replaced with 10 ml of complete medium. The following day, cells expressing high levels of GFP were isolated by FACS (FACScalibur; Becton Dickinson, Mountain View, CA) and replated. Subsequent FACS analysis of the replated cells indicated that >90% of the population was positive for GFP fluorescence. Experiments were then carried out using this population of cells. The control Rh1 cells expressing GFP were prepared as described above except for the substitution of the MSCV-I-GFP vector for the MSCV-I-GFP-RasN17 vector in the production of the pseudotyped virus.
Ras Activation Assay.
We seeded 4 x 106 untransfected Rh1 cells or 4 x 106 Rh1 cells infected with MSCV-I-GFP or MSCV-I-GFP/RasN17 onto 10-cm plates containing RPMI 1640 supplemented with 2 mM L-glutamine and 10% FBS. After the cells were incubated overnight at 37°C to permit attachment to the surface of the plate, the medium was aspirated; after the cells were washed twice with 1x HBSS, 10 ml of RPMI 1640 containing only 2 mM L-glutamine was added. When the confluence reached 90%, the cells were serum-starved for 36 h and then stimulated with IGF-I (10 ng/ml) for 5 min. Cells were quickly chilled and washed with ice-cold PBS. The extent of Ras activation was measured using the Ras Activation Assay kit (Upstate Biotechnology). In microcentrifuge tubes, cells were lysed by the addition of 700 µl of ice-cold 1x MLB buffer. Cell lysates were diluted in MLB to a concentration of 
1 µg/ml total protein, and then 10 µl of glutathione-agarose beads were added to remove protein that would bind to the agarose beads. Samples were stirred by rotation for 25 min at 4°C and centrifuged at 17, 500 x g for 10 min at 4°C. Supernatants were transferred to unused microcentrifuge tubes, and 10 µl (10 µg) of Raf1 RBD-GST conjugated to agarose beads was added. After samples were gently mixed by rotation at 4°C for 30 min, the agarose beads were collected by centrifugation at 17,500 x g for 1 min. Once the supernatant was removed, the beads were washed three times with MLB and resuspended in 25 µl of 2x loading buffer [125 mM Tris (pH 6.8), 4% SDS, 20% glycerol, 100 mM DTT (Bio-Rad), and 0.02% bromphenol blue]. After boiling, the proteins were resolved by electrophoresis in a 12% SDS polyacrylamide gel (Bio-Rad), transferred to a PVDF membrane, and incubated with an anti-Ras antibody (antibody to clone Ras10) to detect activated Ras. Antigen-antibody complexes were detected by using peroxidase-coupled secondary antibodies and enhanced chemiluminescence.
Plasmids and Transfection.
For these experiments, the control vector was pUSEamp+ (Upstate Biotechnology), and the test plasmid was pUSEamp into which cDNA encoding a dominant-negative form of Akt1 (mutation: K179M; Upstate Biotechnology) was cloned. The test plasmid was designated pUSE-dnAkt. Plasmids were isolated and purified by using a plasmid maxi kit from Qiagen (Valencia, CA).
The day before transfection, Rh1 cells (105/well) were plated in six-well plates (well diameter, 35 mm; Falcon; Becton Dickinson Labware, Franklin, NJ) containing RPMI 1640. Once the monolayer was 80% confluent, the cells were transfected with pUSEamp+ or pUSE-dnAkt by using the FuGENE6 transfection reagent according to the manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN). The optimal FuGENE6:DNA ratio was 6:1. After 72 h in culture, the transfected cells were trypsinized, cultured in the same medium supplemented with 500 µg/ml geneticin (Life Technologies, Inc. Rockville, MD), and cloned.
Characterization of Rh1 Cells That Express Dominant-Negative Akt.
The expression of dominant-negative Akt in each clone was monitored by immunoprecipitation and Western blot analysis of cellular c-Myc-tagged Akt protein. Untransfected Rh1 cells and Rh1 cells transfected with pUSE or pUSE-dnAkt cells were seeded at a density of 3 x 106/10-cm plate in RPMI 1640 and incubated until the monolayer was 90% confluent. Cells were washed twice with ice-cold PBS and lysed in 500 µl of RIPA buffer on ice. After the cells were subjected to sonication for 3 s, the resulting lysates were centrifuged at 17,500 x g for 10 min and transferred to fresh microcentrifuge tubes. The cell lysates were diluted to roughly 1 mg/ml (protein) with RIPA buffer, and 20 µl of protein A/G PLUS Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and 2 µl of zysorbin were added to the lysates. Samples were gently mixed by rotation at 4°C for 1 h and then centrifuged for 5 min at 17,500 x g; supernatants were transferred to fresh tubes. Five µg (25 µl) of mouse monoclonal IgG1 anti-c-Myc (9E10) antibody (Santa Cruz Biotechnology) were added to cell lysates, and samples were mixed by rotation for 3 h at 4°C. Then, 30 µl of protein A/G PLUS Agarose beads were added, and the samples were mixed by rotation at 4°C overnight. After centrifugation, the beads were washed three times with ice-cold PBS, resuspended in 30 µl of 1x loading buffer, boiled, and centrifuged. Proteins were resolved by electrophoresis in a 12% SDS polyacrylamide gel, transferred to a PVDF membrane, and incubated with a rabbit polyclonal anti-Akt antibody (Cell Signaling Technology) to detect the c-Myc-tagged Akt.
In Vitro Akt Kinase Assay.
Untransfected Rh1 cells or Rh1 cells transfected with pUSE or pUSE-dnAkt were seeded in MN2E medium at a density of 3 x 106/10-cm plate. After 24 h, cells were stimulated with IGF-I (10 ng/ml) for 10 min. Cells were washed once with ice-cold PBS. We then used the Akt Kinase Assay kit (Cell Signaling Technology) according to the manufacturer’s instructions to analyze the amount of activated Akt present.
Briefly, cells were lysed in 200 µl of ice-cold 1x lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium PPi, 1 mM ß-glycerol phosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1 mM leupeptin] and incubated for 10 min on ice. The cell lysates were centrifuged for 10 min at 17,500 x g at 4°C. The volumes of the supernatants were adjusted so that each sample contained an equal amount of protein (150 µg); the supernatants were then incubated with immobilized (cross-linked) anti-Akt antibody for 3 h at 4°C. The immunoprecipitates were pelleted, and washed twice in ice-cold cell lysis buffer and twice in kinase buffer [25 mM Tris (pH 7.5), 5 mM ß-glycerol phosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2]. The pellets were suspended in 40 µl of kinase buffer containing 200 µM ATP and 1 µg of a GSK-3 fusion protein (CGPKGPGRRGRRRTSSFAEG), which served as the substrate. After the suspensions were incubated at 30°C for 30 min, the reaction was terminated by the addition of 3x SDS sample buffer [187.5 mM Tris-HCl (pH 6.8), 6% SDS, 30% glycerol, 150 mM DTT, and 0.03% bromphenol blue]. The samples were boiled for 5 min, and the proteins were separated on a 12% SDS polyacrylamide gel and subsequently transferred to a PVDF membrane. Membranes were incubated with rabbit polyclonal anti-phospho-GSK-3/ß (Ser21/9) antibody. The kinase assay was repeated three times.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The addition of IGF-I or insulin substantially reduced the apoptotic subpopulation to about 10% of the entire population. In contrast, EGF and PDGF showed only marginal effects on viability. Rapamycin treatment resulted in 80% of cells undergoing apoptosis. Cells exposed to rapamycin appeared to lose membrane integrity late in apoptosis yet remained attached to the culture dish. Addition of IGF-I or insulin resulted in essentially complete protection from apoptosis, whereas EGF increased the proportion of viable rapamycin-treated cells from 21% to 50%; PDGF increased the proportion to 58%. To demonstrate rapamycin treatment inhibited mTOR activity under these growth conditions the phosphorylation status of a downstream substrate, p70S6K, was examined after treatment with rapamycin or inhibitors of PI3K. As shown in Fig. 1
, 2-h pretreatment with rapamycin (100 ng/ml) or the PI3K inhibitors, wortmannin (0.93 µM) and LY294002 (20 µM), completely blocked IGF-I-stimulated activation of p70S6K. Consistent with this data, we have shown previously that IGF-I does not reactivate mTOR in rapamycin-treated cells (42)
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. IGF-I signaling was completely inhibited by 3 µM of PD 98059. To determine how quickly PD 98059 inhibited IGF-I activation of Erk1 and Erk2, we exposed cells grown in MN2E medium to 15 µM of PD 98059 and incubated the cells for different periods of time (up to 120 min). At each time point, cells were stimulated with IGF-I for 5 min, and the phosphorylation of Erk1and Erk2 was evaluated (Fig. 2B)
. Our results indicated that an incubation time of 30 min was sufficient to completely abrogate the IGF-I-induced phosphorylation of Erk1 and Erk2. To determine whether this phosphorylation was inhibited for prolonged periods, Rh1 cells were grown for 1 or 6 days in the presence of PD 98059 (15 µM) and then subjected to IGF-I treatment (10 ng/ml) for 5 min. IGF-I-stimulated phosphorylation of the two proteins in cells grown in PD 98059 for 6 days was markedly (95%) inhibited (Fig. 2C)
. Therefore, pretreatment of cells with PD 98059 inhibited IGF-I-dependent activation of Erk1 and Erk2 in a dose- and time-dependent manner.
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17% when cells were treated with IGF-I (Table 2)
. PD 98059 alone increased the fraction of cell scored as apoptotic to 56%, and this limited apoptosis was prevented by IGF-I (80% viability). In contrast, rapamycin or PD98059 combined with rapamycin induced a significant increase in the proportion of apoptotic cells (78% apoptosis). IGF-I completely abrogated apoptosis of cells treated with rapamycin alone or almost completely with rapamycin plus PD98059 (76% viability). Therefore, under conditions in which PD98059 inhibited MEK1 signaling, IGF-I protected cells from rapamycin-induced apoptosis. These results suggest that activation of Erk1 and Erk2 is not required for IGF-I-mediated protection from rapamycin-induced apoptosis.
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. These results suggest that Ras activation was completely suppressed by the dominant-negative RasN17. Western blot analysis using the same anti-Ras antibody as was used in the activated Ras assays showed that cells expressing dominant-negative RasN17 have higher levels of Ras than Rh1 cells that are uninfected or are infected with the control vector.
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, top panel). Thus, results obtained with this dominant-negative Ras are in contrast to the results obtained with the pharmacological inhibitor of farnesyltransferase (above). PD 98059 treatment (15 µM), which was used as a positive control, resulted in the complete blockade of the ability of the growth factor to activate Erk1 or Erk2 in Rh1 cells alone and in Rh1 cells infected with MSCV-I-GFP. Unlike its inhibition of IGF-I-mediated activation of Erk1 and Erk2, RasN17 did not inhibit IGF-I-mediated activation of Akt (Fig. 3C)
. These findings indicate that Akt is not a downstream target of Ras in the IGF-I signaling pathway of Rh1 cells.
IGF-I Inhibits Rapamycin-induced Apoptosis of Rh1 Cells That Overexpress RasN17.
Uninfected Rh1 cells and those stably expressing MSCV-I-GFP/RasN17 were preincubated with or without rapamycin (100 ng/ml) for 4 days. Duplicate samples were coincubated with or without IGF-I (10 ng/ml). The percentage of apoptotic cells within each population was then evaluated. In populations of Rh1 cells and those expressing GFP/RasN17, both of which were grown in MN2E medium, 17% of the cells were apoptotic (Table 3)
. Rapamycin treatment for 4 days substantially increased the proportion of apoptotic cells in the populations expressing GFP/RasN17 (40%). IGF-I treatment of cell populations expressing GFP/RasN17 decreased the percentage of apoptotic cells to their respective control levels. The level of necrotic cells was similar (
2%) in both populations of Rh1 cells and those expressing GFP/RasN17 cells under these conditions. Results for Rh1 cells expressing only GFP were similar to parental Rh1 cells (data not shown). These results clearly suggest that suppression of apoptosis by IGF-I is not Ras-dependent.
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. Akt activation was evident after 5 min of treatment with any of the three growth factors. Stimulation by EGF was transient and returned to the basal levels within 30 min. In contrast, phosphorylation of Akt by PDGF was more prolonged, and Akt phosphorylation by IGF-I remained stable for at least 2 h. The total amount of Akt was unchanged after growth factor stimulation. These results indicate that IGF-I, EGF, and PDGF receptors mediate activation of the Akt pathway in Rh1 cells.
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IGF-I Prevents Rapamycin-induced Apoptosis of Rh1 Cells Treated with LY 294002.
To determine whether the effect of IGF-I on inhibition of rapamycin-induced apoptosis involved the PI3K pathway, we treated Rh1 cells grown in MN2E medium with DMSO (0.1%), rapamycin (100 ng/ml), LY 294002 (20 µM), or rapamycin (100 ng/ml) and LY 294002 (20 µM). Duplicate samples were coincubated with or without IGF-I (10 ng/ml). Our assessment of apoptosis indicated that 23% of the untreated cell populations consisted of annexin V-positive and propidium iodide-positive cells, but IGF-I reduced this proportion to 15% (Table 4)
. LY 294002 treatment resulted in apoptosis of 54% of cells. When the cells were exposed to LY 294002 and rapamycin, only 33% of cells were scored as viable. Coincubation with IGF-I almost completely protected cells under all conditions of drug exposure. Thus, concentrations of LY 294002 that markedly inhibit PI3K activity failed to blunt the antiapoptotic effect of IGF-I. Moreover, IGF-I prevented apoptosis induced by these agents either alone or in combination with rapamycin. IGF-I also partially rescued from higher concentrations of LY294002 (50 and 100 µM) that completely abrogate PI3K signaling. Thus, at 100 µM of LY294002 IGF-I increased the proportion of viable cells from 19% to 59%. Qualitatively similar results were obtained using the structurally distinct PI3K inhibitor wortmannin (data not shown). Because Akt is an effector of survival signaling and is downstream of PI3K, our results can be explained in two ways: (a) Akt may be activated in a PI3K-independent fashion; or (b) an Akt-independent pathway may be used. If the first explanation were correct Akt activation induced by IGF-I would not be inhibited by LY 294002. However, IGF-I-mediated activation of endogenously expressed Akt was sensitive to PI3K inhibitors, a result that favors the existence of an Akt-independent pathway in Rh1 cells.
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, the anti-c-Myc antibody was used to immunoprecipitate the tagged protein, which was then subjected to immunoblotting analysis with an anti-Akt antibody. The Akt band was detected in cells transfected with the dominant-negative Akt expression plasmid but not in parental Rh1 cells or in cells transfected with control vector (pUSE) alone (Fig. 5A)
. To confirm that this dominant-negative Akt protein inhibited the activation of endogenous Akt, we stimulated untransfected Rh1 cells, Rh1 cells transfected with pUSE, and Rh1 cells transfected with pUSE-dnAkt with IGF-I (10 ng/ml) for 10 min, and then lysed the cells on ice. Activation of Akt by IGF-I was evaluated by assessing the phosphorylation of Ser473 of Akt or the in vitro kinase activity of protein immunoprecipitated by the anti-Akt antibody. IGF-I stimulated phosphorylation of Ser473 in parental Rh1 cells and Rh1 cells transfected with pUSE, but not in those transfected with pUSE/dnAkt (Fig. 5B
, top panel). After the membrane was stripped of bound antibody, the membrane was incubated with the anti-Akt antibody to determine the total amount of Akt in each cell line (Fig. 5B
, center panel). The total amount of Akt was 4-fold greater in Rh1 cells transfected with pUSE/dnAkt than in parental or vector-transfected cells.
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, bottom panel). These results indicate that the dominant-negative Akt effectively blocks the activity of endogenous Akt.
IGF-I-mediated Protection of Rh1 Cells From Rapamycin-induced Apoptosis Is Independent of Akt Signaling.
To determine whether IGF-I-mediated inhibition of rapamycin-induced apoptosis is independent of Akt signaling, we exposed cells expressing dominant-negative Akt to rapamycin (100 ng/ml) for 4 days in the presence or absence of IGF-I (10 ng/ml), and then determined the extent of apoptosis within each treatment population. The level of viability was slightly lower in this experiment: 35% of cells within the control population were both annexin V- and propidium iodide-positive (Fig. 6)
. IGF-I reduced this level to 14–19%. When the cells transfected with either pUSE-dnAkt or pUSE were treated with rapamycin, the proportion of apoptotic cells increased to 79% and 75%, respectively. Addition of IGF-I completely protected cells expressing dominant-negative Akt from rapamycin-induced apoptosis. This finding suggests that the protection is independent of PI3K and Akt activity.
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10% of untreated cells were apoptotic (Fig. 7)
. Approximately 20% of the population treated with only rapamycin were apoptotic. In contrast, treatment with LY 294002 alone resulted in 53% apoptosis; and PD 98059, 13%. Treatment with rapamycin and each of the other signaling inhibitors resulted in essentially additive levels of apoptosis. IGF-I (10 ng/ml) completely inhibited apoptosis induced by rapamycin in the presence or absence of the other signaling inhibitors (Fig. 7)
. Taken together these results additionally support our findings that IGF-I prevents rapamycin-induced apoptosis of Rh1 cells in a Ras-MEK-Erk1-Erk2/PI3K-independent manner.
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25% of untreated cells were apoptotic (Table 5)
. Treatment with LY 294002 or PD 98059 resulted in apoptosis of 58% and 22% of cells, respectively. However, LY 294002 or PD 98059 in combination with rapamycin increased the proportion of apoptotic cells to 69% and 35%, respectively. Important is the finding that IGF-I prevented apoptosis caused by these agents either separately or in combination.
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. IGF-I partially rescued cells from apoptosis induced by LY294002 alone or in combination with rapamycin. IGF-I also rescued cells from apoptosis induced by PD98059 as a single agent, or when combined with rapamycin and exposed for 6 days (Table 7)
. Furthermore, IGF-I prevented apoptosis induced when Rh30 cells were exposed to PD98059 combined with LY98059 for 45 h (Fig. 8)
. The combination of PD98059 plus LY294002 resulted in 78% loss of viability, whereas in the presence of IGF-I this was reduced to 33%.
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DISCUSSION |
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The MAPK pathway is responsible for mediating numerous effects of IGF-I (46
, 47)
in part through a Ras-dependent activation of Raf/MEK1 leading to activation of Erk1 and Erk2. Prolonged inhibition of Erk1/2 activation by PD 98059, a specific noncompetitive inhibitor of MEK1, with respect to ATP binding (43)
, in the presence of rapamycin induced 75% apoptosis that was almost completely abrogated by IGF-I. Thus, our results demonstrate that MEK1 activation of Erk1/2 MAPK pathway is not essential for IGF-I protection from rapamycin-induced apoptosis. Similar results were obtained in Rh30 cells.
We next examined whether Ras signaling was necessary for IGF-I-mediated protection. Ras signaling was abrogated genetically by infecting Rh1 cells with a retrovirus carrying a dominant-negative mutant of Ras (RasN17). The dominant-negative RasN17 was expressed at high levels for at least 5 days after infection of Rh1 cells and inhibited Ras activation by IGF-I. RasN17 suppressed Erk-1/2 phosphorylation completely, suggesting that SCH66336 incompletely inhibits this pathway in Rh1 cells. Importantly, IGF-I completely protected the RasN17 expressing cells from rapamycin-induced apoptosis. Taken together, these data appear to confirm that Ras signaling is not required for IGF-I to protect against rapamycin-induced apoptosis. Our data also indicate that Ras is not upstream of Akt, as phosphorylation of Akt by IGF-I was not inhibited in RasN17-expressing cells. Hence, these results differ from reports implicating Ras/Akt signaling in survival (48) .
We next focused on the role of PI3K-mediated activation of Akt in suppression of apoptosis (49 , 50) . The protective effect of IGF-I may result from the phosphorylation of both proapoptotic proteins (19 , 48 , 51) and transcriptional targets involved in cell survival (23 , 45 , 52) . Akt requires the successive action of lipid and protein kinases for activation (53 , 54) . Because PI3K is known to activate Akt we first determined the effect of IGF-I, EGF, and PDGF on Akt activation. IGF-I treatment of cells resulted in a sustained phosphorylation of Akt, whereas after stimulation by EGF phosphorylation declined within 30 min to basal level. The effect of PDGF was intermediate. Thus, there was some correlation with the ability of these growth factors to protect cells from rapamycin-induced apoptosis. As anticipated, activation of Akt by IGF-I, EGF, or PDGF was inhibited by LY 294002. The ability of this PI3K inhibitor to decrease Akt phosphorylation suggests that the activation of Akt is PI3K-dependent, although we have not directly assayed PI3K activity in Rh1 cells. In reported studies (55 , 56) IGF-I protection of cells from apoptosis required activation of PI3K, and activation of Akt was sufficient for protection (32) .
Because of the potential breakdown of LY294002, assays for apoptosis were limited to 24–45 h exposures. LY 294002 with or without rapamycin for 24 h increased the proportion of apoptotic cells to 54 and 67%, respectively, but addition of IGF-I could completely overcome this effect and protect the cells from death. Thus, the concentration of LY 294002 that produced very marked inhibition of PI3K signaling was unable to attenuate the antiapoptotic effect of IGF-I. Apoptosis at higher concentrations of LY294002 that completely inhibit PI3K signaling was also significantly inhibited by IGF-I. However, apoptosis caused by such drug concentrations may be in part independent of the inhibition of PI3K. However, our results indicate that rescue by IGF-I from rapamycin-induced apoptosis is predominantly independent of the PI3K/Akt signaling pathway.
To additionally test whether PI3K/Akt pathway was required for IGF-I-induced cell survival, we used a genetic approach. Expression of the dominant-negative Akt significantly inhibited phosphorylation of Akt (Ser473) and endogenous Akt kinase activity in response to IGF-I stimulation, thus demonstrating function. However, massive apoptosis (
75%) induced by rapamycin was completely protected by IGF-I in Rh1/dnAkt cells. Thus, Akt activity is not required for survival mediated by IGF-I. The PI3K inhibitor also caused a high level of apoptosis (60%) in cells expressing dnAkt. This suggests that Akt is not the major downstream target of PI3K involved in cell survival in these cells.
There is accumulating evidence indicating various levels of cross-talk between the MAPK and the PI3K/Akt pathways, which may play a role in regulating apoptosis. Thus, it is possible that Ras and PI3K/Akt pathways could be redundant, and that blocking each individually still allows IGF-I-mediated protection from apoptosis. Therefore, we have investigated whether IGF-I could still protect cells from apoptosis after simultaneously blocking these two major signaling pathways by genetic and pharmacological approaches. IGF-I protected against rapamycin-induced apoptosis in Rh1 cells expressing dominant-negative RasN17 in the presence of PI3K inhibitor, MEK1 inhibitor, or both agents. Similarly, IGF-I completely protected Rh1 cells expressing dominant-negative Akt exposed to rapamycin, LY 294002, and PD 98059. Thus, simultaneous inhibition of both PI3K/Akt and Ras signaling pathways did not abrogate the protective action of IGF-I. Similarly, IGF-I significantly protected Rh30 cells treated with a combination of LY294002 and PD98059. Taken together, these data additionally support the contention that neither the MAPK nor PI3K/Akt-dependent pathways are required for IGF-I to protect against rapamycin-induced apoptosis.
Our studies have focused on determining whether signaling through PI3K/Akt or Ras/Erk1/2 is involved in IGF-I-mediated protection of rapamycin-induced apoptosis. Survival in Rh1 and Rh30 cells appears independent of either pathway. IGF-I can activate other MAPKs, such as c-Jun NH2-terminal kinase or p38, and these pathways have not been studied. Consequently, additional studies are needed to identify the signaling pathway(s) by which IGF-I protects Rh1 cells from apoptosis. As mTOR inhibition by rapamycin mimics certain effects of amino acid or glucose deprivation (57 , 58) , it is possible that the effects of IGF-I or insulin on nutrient regulation may play a role in protection from apoptosis. The identification and characterization of these novel pathways and the development of inhibitors of these novel pathways may be important for enhancing the cytotoxic effect of agents that target mTOR, such as CCI-779 and the RAD001 rapamycin analogs that are currently in Phase I/II trials as cancer treatment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by USPHS awards CA77776, CA23099, and CA28765 (Cancer Center Support Grant) from the National Cancer Institute; by a grant from Wyeth-Ayerst Laboratories; and by the American Lebanese Syrian Associated Charities (ALSAC). 
2 To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Mail Stop 230, 332 North Lauderdale Street, Memphis, TN 38105-2794. Phone: (901) 495-3440; Fax: (901) 495-4290; E-mail: peter.houghton{at}stjude.org
3 The abbreviations used are: IGF-I, insulin-like growth factor; ERK, extracellular signal-regulated kinase; RBD, Ras-binding domain; GFP, green fluorescent protein; RIPA, radioimmunoprecipitation assay; PDK, phosphoinositide-dependent kinase; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; IGF-IR, insulin-like growth factor I receptor; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3'-kinase; EGF, epidermal growth factor; PDGF; platelet-derived growth factor; GSK-3, glycogen synthase kinase 3; MN2E, modified N2E medium; FACS, fluorescence-activated cell sorting; PVDF, polyvinylidene difluoride; GST, glutathione S-transferase.
Received 4/17/02. Accepted 11/13/02.
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REFERENCES |
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