[CANCER RESEARCH 63, 364–374, January 15, 2003]

Insulin-like Growth Factor I-mediated Protection from Rapamycin-inducedApoptosis Is Independent of Ras-Erk1-Erk2 and Phosphatidylinositol3�-Kinase-Akt Signaling Pathways1

Kuntebommanahalli N. Thimmaiah, John Easton, Shile Huang, Karen A. Veverka, Glen S. Germain,Franklin C. Harwood, and Peter J. Houghton2

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

ABSTRACT

The mTOR inhibitor rapamycin induces G1 cell cycle accumulation andp53-independent apoptosis of the human rhabdomyosarcoma cell lineRh1. Insulin-like growth factor I (IGF-I) and insulin, but not epidermalgrowth factor or platelet-derived growth factor, completely preventedapoptosis of this cell line. Because the Ras-Erk1-Erk2 and phosphatidy-linositol 3�-kinase (PI3K)-Akt pathways are implicated in the survival ofvarious cancer cells, we determined whether protection from rapamycin-induced apoptosis by IGF-I requires one or both of these pathways.Despite the blocking of Ras-Erk signaling by the addition of PD 98059 (aMEK1 inhibitor) or by the overexpression of dominant-negative RasN17,IGF-I completely prevented rapamycin-induced death. Inhibition of Rassignaling did not prevent Akt activation by IGF-I. To determine the roleof the PI3K-Akt pathway in rescuing cells from apoptosis caused byrapamycin, cells expressing dominant-negative Akt were tested. Thismutant protein inhibited IGF-I-induced phosphorylation of Akt andblocked phosphorylation of glycogen synthase kinase 3. The preventionof rapamycin-induced apoptosis by IGF-I was not inhibited by expressionof dominant-negative Akt either alone or under conditions in whichLY 294002 inhibited PI3K signaling. Furthermore, IGF-I preventedrapamycin-induced apoptosis when the Ras-Erk1-Erk2 and PI3K-Aktpathways were blocked simultaneously. Similar experiments in a secondrhabdomyosarcoma cell line, Rh30, using pharmacological inhibitors ofPI3K or MEK1, alone or in combination, failed to block IGF-I rescuefrom rapamycin-induced apoptosis. Therefore, we conclude that a novelpathway(s) is responsible for the IGF-I-mediated protection against ra-pamycin-induced apoptosis in these rhabdomyosarcoma cells.

INTRODUCTION

IGFs3, IGF-I and IGF-II are soluble peptide factors that circulatewhile bound to one of six IGF-binding proteins. The IGFs, theirreceptors, and the IGF-binding proteins constitute a family of cellularmodulators that play essential roles in regulating growth and devel-opment (1). The action of IGFs results primarily from the activationof the IGF-IR (2). This receptor resembles the insulin receptor instructural as well as functional aspects (3), and is a heterotetramerictransmembrane glycoprotein consisting of 2 �- and 2 �-subunits. The

tyrosine kinase catalytic site and the ATP-binding site are located inthe cytoplasmic portion of the �-subunit. The tyrosine kinase activityof the �-subunit is stimulated when IGF-I binds to the �-subunit.Intracellular substrates of the IGF-IR include the insulin receptorsubstrates IRS-1 and IRS-2, and the SH2-containing protein Shc.Phosphorylation of these proteins results in their interaction with othersignaling molecules, such as the adapter Grb2, that are, in turn,coupled to effector molecules, such as the guanine nucleotide ex-change factor Sos (4–6) and PI3K (7, 8). The activated Shc-Grb2-Soscomplex activates the small GTP-binding protein Ras, and activatedRas initiates a cascade of Ser/Thr kinases, some of which eventuallytranslocate to the nucleus to stimulate gene expression (9, 10). Theextracellular signal-regulated MAPKs, Erk1 and Erk2, are key inter-mediates in the propagation of signals from many growth factorreceptors to the nucleus (9, 11). Erk proteins are activated by MEK,a dual function kinase that phosphorylates tyrosine and threonineresidues of Erk (12, 13) downstream of Ras and Raf1.

IRS-1 and IRS-2 lie upstream of a signaling cascade involvingPI3K. Phosphoinositides phosphorylated by PI3K recruit PDKs suchas PDK1 to the plasma membrane. Evidence suggests that phospho-rylation of Thr308 and Ser473 of Akt by PDK1 and PDK2 activatesAkt (14–16). This event results in Akt phosphorylation of its down-stream substrates such as GSK-3 (17), 6-phosphofructo-2-kinase (18),the Bcl2 family member Bad (19), caspase-9 (20), nitric oxide syn-thase (21, 22), and the winged-helix family of FKHRL1 transcriptionfactors (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 thegrowth of cells by inducing mitogenesis and transformation, and byprotecting cells from various apoptotic injuries (26). Several reportshave documented recently the involvement of IGF-I in regulatingapoptosis (27) induced by various stimuli, including physiologicalstress (28), hyperosmosis (29, 30), chemotherapy (31), and DNAdamage caused by chemotherapeutic drugs or UV-B radiation(32–34).

Additional evidence of the role of IGF-I in regulating apoptosis hasbeen provided by studies involving rapamycin, an immunosuppressivemacrocyclic lactone that specifically inhibits the activity of mTOR, aSer/Thr kinase downstream of PI3K. Inhibition of mTOR leads to G1

arrest of many malignant cell lines, and currently analogs of rapamy-cin are being investigated as cancer therapeutic agents. We havereported previously that rapamycin selectively induces apoptosis oftumor cells that express mutant p53 and are grown under serum-freeconditions (35). However, the addition of IGF-I to the growth mediumcompletely protects Rh1 cells from rapamycin-induced apoptosis.Therefore, we are interested in understanding how IGF-I protects Rh1cells 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 ofprotein kinases (4). EGF activates the PI3K-Akt signaling pathway inseveral EGF receptor-overexpressing cells such as prostate cancercells (36), epidermoid cancer cells (37), and ovarian cancer cells (38).

Received 4/17/02; accepted 11/13/02.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by USPHS awards CA77776, CA23099, and CA28765 (CancerCenter Support Grant) from the National Cancer Institute; by a grant from Wyeth-AyerstLaboratories; and by the American Lebanese Syrian Associated Charities (ALSAC).

2 To whom requests for reprints should be addressed, at Department of MolecularPharmacology, St. Jude Children’s Research Hospital, Mail Stop 230, 332 North Lauder-dale Street, Memphis, TN 38105-2794. Phone: (901) 495-3440; Fax: (901) 495-4290;E-mail: peter.houghton@stjude.org.

3 The abbreviations used are: IGF-I, insulin-like growth factor; ERK, extracellularsignal-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, phosphati-dylinositol 3�-kinase; EGF, epidermal growth factor; PDGF; platelet-derived growthfactor; GSK-3, glycogen synthase kinase 3; MN2E, modified N2E medium; FACS,fluorescence-activated cell sorting; PVDF, polyvinylidene difluoride; GST, glutathioneS-transferase.

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Extensive studies have shown that the pathways used for all of thefunctions of IGF-IR appear to overlap considerably. Because theRas-Erk1-Erk2 and PI3K-Akt pathways are the two major implicatedin survival signaling in a wide variety of cancer cells (reviewed in Ref.2), we sought to determine whether one or both of these pathways isrequired for the IGF-I-mediated prevention of rapamycin-inducedapoptosis of Rh1 and Rh30 cells.

MATERIALS AND METHODS

Inhibitors. Rapamycin, a generous gift from James Gibbons (Wyeth-Ayerst, Pearl River, NJ). LY 294002, wortmannin, and PD 98059 werepurchased from Calbiochem (Cambridge, MA).

Cell Lines and Growth Conditions. The human rhabdomyosarcoma celllines Rh1 and Rh30 have been described (35). Briefly, Rh1 and Rh30 cellswere 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, cellmonolayers were washed with RPMI 1640 containing 2 mM L-glutamine andincubated in the same medium. For prolonged serum-free experiments, cellswere cultured in modified N2E (MN2E) medium (DMEM/F-12; 1:1 mixture)supplemented with 1 �g/ml human holo transferrin, 30 nM sodium selenite, 20nM progesterone, 100 �M putrescine, 30 nM vitamin E phosphate, 50 �M

ethanolamine, and 1 mg/ml BSA (Sigma, St. Louis, MO). Cells in MN2Emedium containing 5 �g/ml bovine fibronectin (Sigma) were plated andallowed 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 cellsalone, Rh1 cells transduced with MSCV-I-GFP or MSCV-I-GFP/RasN17, orRh1 cells transfected with pUSE or pUSE-dnAkt (1.5 � 106/162-cm2 flask)were grown overnight in MN2E medium. On day 1, cells were treated withDMSO (0.1%; vehicle control) or rapamycin (100 ng/ml). The appropriategrowth factors recombinant human IGF-I and PDGF (both from UpstateBiotechnology) and the sodium salt of human recombinant insulin and recom-binant human EGF (both from Sigma) were added individually to some of thecells treated with DMSO and to some treated with rapamycin. After 4 days, thecells were trypsinized, washed with PBS, and resuspended in 200 �l of bindingbuffer. Cells were incubated with 10 �l of annexin V-FITC (final concentra-tion, 1 �g/ml) and 500 ng of propidium iodide in a final volume of 410 �l.Cells were incubated at room temperature in the dark for 10 min before flowcytometric analysis (FACScalibur; Becton Dickinson) was performed as de-scribed (35). Statistical significance of differences between viable cells incontrol and treatment groups was tested using the Student unpaired t test.

Western Blot Analysis. Cultured cells were briefly washed with ice-coldPBS. For the analysis of Akt, Erk1, and Erk2, cells were placed on ice andlysed 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 phenylmeth-ylsulfonyl fluoride, 5 �g/ml aprotinin, and 5 �g/ml leupeptin. For the detectionof 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 werelysed 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 �g/ml aprotinin,1 �g/ml leupeptin, one Complete mini protease inhibitor tablet (BoehringerMannheim, Mannheim, Germany), 1 mM phenylmethylsulfonyl fluoride, 1 mM

Na3VO4, and 1 mM NaF]. Cellular debris was pelleted by centrifugation at17,500 � g for 10 min at 4°C. Protein concentration of the supernatants wasmeasured by the bicinchoninic acid assay using BSA as the standard (Pierce,Rockford, IL).

For the analysis of Akt, p70S6K, Erk1, Erk2, and Ras, equivalent amountsof proteins from various samples were subjected to electrophoresis through a12% SDS-polyacrylamide gel (Bio-Rad) and subsequently transferred toPVDF membranes (Immobilon; Millipore, Bedford, MA). After a 1-h incuba-tion in 1� Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat drymilk at room temperature, the wet PVDF membranes were then incubated withthe appropriate antibody: rabbit polyclonal antiserum specific for the phos-phorylated Thr202 of Erk1 and the phosphorylated Tyr204 of Erk2 (dilution,1:4,000; Cell Signaling Technology, Beverly, MA); rabbit polyclonal anti-

serum specific for the phosphorylated Ser473 of Akt (dilution 1:4,000; CellSignaling Technology); rabbit polyclonal antiserum specific for the phospho-rylated Thr389 of p70S6K (dilution 1:4,000; Cell Signaling Technology); andmouse monoclonal antibody (IgG2a�) to Ras or Ras10 (dilution, 1:1,000;Upstate Biotechnology, Lake Placid, NY). The secondary antibody was eitherthe 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 tovisualize bound antibody.

To ensure that equivalent amounts of protein were loaded on each gel, all ofthe immunoblots were treated with stripping buffer [62.5 mM Tris-HCl (pH6.7), 2% SDS, and 100 mM �-mercaptoethanol] for 30 min at 50°C and thenincubated with one of the appropriate antibodies: rabbit polyclonal antibody toErk2 (K-23; dilution, 1:4000; Upstate Biotechnology), rabbit polyclonal anti-body to Akt (dilution, 1:4000; Cell Signaling Technology), or mouse mono-clonal antibody to �-tubulin (dilution, 1:2000; Sigma). The secondary anti-bodies and detection of bound antibody were as described in the precedingparagraph.

RasN17 Retrovirus. The DNA sequence coding for the dominant-negativeform of Ras, RasN17 (39), was subcloned into the EcoRI site of the retroviralvector MSCV-I-GFP (40), which contains an internal ribosomal entry site fortranslation of the single transcript encoding both GFP and RasN17. Viralparticles pseudotyped with the feline endogenous virus (RD114) envelopeprotein were then generated by transiently transfected 293T cells (41). Briefly,15 �g of the helper vector PAM3E�, 12.5 �g of MSCV-I-GFP-RasN17, and3 �g of the helper vector RD114 were transfected into 3 � 106 293T cells ona 10-cm plate by using the N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonicacid/calcium method. After 12 h, the medium was replaced with fresh DMEMsupplemented with 10% FBS. After an additional 4 h, the medium wasreplaced with 3 ml of complete DMEM. Thereafter, at the end of every 6 hduring a 48-h period, the medium was collected and placed on ice. Thecollected samples were pooled, filtered, and dispensed into aliquots that werestored 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 � 105 infectious particles per ml.

Rh1 cells were transduced with 3 ml of virus in the presence of 10 �g/mlof hexadimethrine bromide (Sigma). After 3 h, the viral supernatant wasreplaced with 10 ml of complete medium. The following day, cells expressinghigh levels of GFP were isolated by FACS (FACScalibur; Becton Dickinson,Mountain View, CA) and replated. Subsequent FACS analysis of the replatedcells indicated that �90% of the population was positive for GFP fluorescence.Experiments were then carried out using this population of cells. The controlRh1 cells expressing GFP were prepared as described above except for thesubstitution of the MSCV-I-GFP vector for the MSCV-I-GFP-RasN17 vectorin the production of the pseudotyped virus.

Ras Activation Assay. We seeded 4 � 106 untransfected Rh1 cells or4 � 106 Rh1 cells infected with MSCV-I-GFP or MSCV-I-GFP/RasN17 onto10-cm plates containing RPMI 1640 supplemented with 2 mM L-glutamine and10% FBS. After the cells were incubated overnight at 37°C to permit attach-ment to the surface of the plate, the medium was aspirated; after the cells werewashed twice with 1� HBSS, 10 ml of RPMI 1640 containing only 2 mM

L-glutamine was added. When the confluence reached 90%, the cells wereserum-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 Rasactivation was measured using the Ras Activation Assay kit (Upstate Biotech-nology). In microcentrifuge tubes, cells were lysed by the addition of 700 �lof ice-cold 1� MLB buffer. Cell lysates were diluted in MLB to a concen-tration of �1 �g/ml total protein, and then 10 �l of glutathione-agarose beadswere added to remove protein that would bind to the agarose beads. Sampleswere stirred by rotation for 25 min at 4°C and centrifuged at 17, 500 � g for10 min at 4°C. Supernatants were transferred to unused microcentrifuge tubes,and 10 �l (10 �g) of Raf1 RBD-GST conjugated to agarose beads was added.After samples were gently mixed by rotation at 4°C for 30 min, the agarosebeads were collected by centrifugation at 17,500 � g for 1 min. Once thesupernatant was removed, the beads were washed three times with MLB andresuspended in 25 �l of 2� loading buffer [125 mM Tris (pH 6.8), 4% SDS,20% glycerol, 100 mM DTT (Bio-Rad), and 0.02% bromphenol blue]. Afterboiling, the proteins were resolved by electrophoresis in a 12% SDS poly-acrylamide gel (Bio-Rad), transferred to a PVDF membrane, and incubatedwith an anti-Ras antibody (antibody to clone Ras10) to detect activated Ras.

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Antigen-antibody complexes were detected by using peroxidase-coupled sec-ondary antibodies and enhanced chemiluminescence.

Plasmids and Transfection. For these experiments, the control vector waspUSEamp� (Upstate Biotechnology), and the test plasmid was pUSEamp intowhich 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 fromQiagen (Valencia, CA).

The day before transfection, Rh1 cells (105/well) were plated in six-wellplates (well diameter, 35 mm; Falcon; Becton Dickinson Labware, Franklin,NJ) containing RPMI 1640. Once the monolayer was 80% confluent, the cellswere transfected with pUSEamp� or pUSE-dnAkt by using the FuGENE6transfection reagent according to the manufacturer’s instructions (Roche Mo-lecular Biochemicals, Indianapolis, IN). The optimal FuGENE6:DNA ratiowas 6:1. After 72 h in culture, the transfected cells were trypsinized, culturedin the same medium supplemented with 500 �g/ml geneticin (Life Technol-ogies, 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 byimmunoprecipitation and Western blot analysis of cellular c-Myc-tagged Aktprotein. Untransfected Rh1 cells and Rh1 cells transfected with pUSE orpUSE-dnAkt cells were seeded at a density of 3 � 106/10-cm plate in RPMI1640 and incubated until the monolayer was 90% confluent. Cells were washedtwice with ice-cold PBS and lysed in 500 �l of RIPA buffer on ice. After thecells were subjected to sonication for 3 s, the resulting lysates were centrifugedat 17,500 � g for 10 min and transferred to fresh microcentrifuge tubes. Thecell lysates were diluted to roughly 1 mg/ml (protein) with RIPA buffer, and20 �l of protein A/G PLUS Agarose (Santa Cruz Biotechnology, Santa Cruz,CA) and 2 �l of zysorbin were added to the lysates. Samples were gentlymixed by rotation at 4°C for 1 h and then centrifuged for 5 min at 17,500 � g;supernatants were transferred to fresh tubes. Five �g (25 �l) of mousemonoclonal 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 �l of protein A/G PLUS Agarose beads were added, and the sampleswere mixed by rotation at 4°C overnight. After centrifugation, the beads werewashed three times with ice-cold PBS, resuspended in 30 �l of 1� loadingbuffer, boiled, and centrifuged. Proteins were resolved by electrophoresis in a12% SDS polyacrylamide gel, transferred to a PVDF membrane, and incubatedwith a rabbit polyclonal anti-Akt antibody (Cell Signaling Technology) todetect the c-Myc-tagged Akt.

In Vitro Akt Kinase Assay. Untransfected Rh1 cells or Rh1 cells trans-fected with pUSE or pUSE-dnAkt were seeded in MN2E medium at a densityof 3 � 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 AktKinase Assay kit (Cell Signaling Technology) according to the manufacturer’sinstructions to analyze the amount of activated Akt present.

Briefly, cells were lysed in 200 �l of ice-cold 1� 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 �-glycerol phosphate, 1 mM Na3VO4, 1 mM phenylmeth-ylsulfonyl fluoride, and 1 mM leupeptin] and incubated for 10 min on ice. Thecell lysates were centrifuged for 10 min at 17,500 � g at 4°C. The volumes of

the supernatants were adjusted so that each sample contained an equal amountof protein (150 �g); the supernatants were then incubated with immobilized(cross-linked) anti-Akt antibody for 3 h at 4°C. The immunoprecipitates werepelleted, and washed twice in ice-cold cell lysis buffer and twice in kinasebuffer [25 mM Tris (pH 7.5), 5 mM �-glycerol phosphate, 2 mM DTT, 0.1 mM

Na3VO4, and 10 mM MgCl2]. The pellets were suspended in 40 �l of kinasebuffer containing 200 �M ATP and 1 �g of a GSK-3 fusion protein (CGP-KGPGRRGRRRTSSFAEG), which served as the substrate. After the suspen-sions were incubated at 30°C for 30 min, the reaction was terminated by theaddition of 3� 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 wereboiled for 5 min, and the proteins were separated on a 12% SDS polyacryl-amide gel and subsequently transferred to a PVDF membrane. Membraneswere incubated with rabbit polyclonal anti-phospho-GSK-3�/� (Ser21/9) an-tibody. The kinase assay was repeated three times.

RESULTS

Effect of Growth Factors on Rapamycin-induced Apoptosis ofRh1 Cells. As a starting point for our investigation to determinewhich growth factors serve as survival factors for Rh1 cells, weexamined the ability of IGF-I, insulin, EGF, and PDGF to protectthese cells from rapamycin-induced apoptosis. Under serum-freegrowth conditions, rapamycin caused a concentration-dependent in-hibition of proliferation (IC50 � 1 ng/ml) and time-dependent apo-ptosis of Rh1 cells. To determine concentrations of growth factorsrequired for maximal protection from apoptosis, we incubated Rh1cells for 6 days in the absence or presence of rapamycin (100 ng/ml)in MN2E medium with increasing concentrations of IGF-I (0.25 to100 ng/ml), insulin (5 to 10,000 ng/ml), EGF (5 to 1,000 ng/ml), orPDGF (5 to 300 ng/ml). Increasing concentrations of the growthfactors resulted in a concentration-dependent protection from rapa-mycin-induced apoptosis. Maximal protection was achieved at 10ng/ml for IGF-I, 250 ng/ml for insulin, 25 ng/ml for EGF, and 25ng/ml for PDGF (data not shown). All of the subsequent experimentswere performed with these concentrations of growth factors to ensuremaximal protection. To quantitate growth factor protection from ra-pamycin-induced apoptosis, Rh1 cells were grown in MN2E mediumand exposed to 0.1% DMSO (vehicle control) or rapamycin (100ng/ml). Some of the cells treated with DMSO and with rapamycinreceived exogenous IGF-I (10 ng/ml), insulin (250 ng/ml), EGF (25

Fig. 1. Rapamycin, and PI3K inhibitors block IGF-I stimulation of p70S6K phospho-rylation. Serum-starved Rh1 cells were treated for 2 h with drug vehicle (0.1% DMSO),rapamycin (100 ng/ml), wortmannin (0.93 �M), or LY294002 (20 �M) at the indicatedconcentrations. Cells were unstimulated or stimulated with IGF-I (10 ng/ml), and activa-tion of p70S6K determined after 10 min. Results show phosphorylated (Thr389) and totalp70S6K. Membranes were stripped and reprobed for �-tubulin as a loading control.

Table 1 Effect of growth factors on rapamycin-induced apoptosis of Rh1 cellsa

Treatmentb

Percentage of cellsa

Viable Apoptotic

Control 72 29IGF-I 89 11Insulin 90 10EGF 74 25PDGF 78 21Rapamycin 21c 78Rapamycin � IGF-I 85 15Rapamycin � insulin 88 12Rapamycin � EGF 50 50Rapamycin � PDGF 58 42

a Results show mean values from two independent experiments.b IGF-I, 10 ng/ml; Insulin, 250 ng/ml; EGF, 25 ng/ml; PDGF, 25 ng/ml; Rapamycin,

100 ng/ml. Necrosis, Annexin V-negative, propidium iodide-positive: �0.7%.c P � 0.05

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ng/ml), or PDGF (25 ng/ml) for 4 days; the remaining cells receivedno growth factors. Cells were harvested, and the extent of apoptosiswas evaluated by the ApoAlert flow cytometric assay. Within theapoptotic population, cells in the early stages of apoptosis wereannexin V-positive and propidium iodide-negative, whereas those inthe late stages were annexin V-positive and propidium iodide-posi-tive. These populations are combined in the tables presented. Approx-imately 30% of cells in the control population were undergoingspontaneous apoptosis at the time of evaluation (Table 1). The addi-tion of IGF-I or insulin substantially reduced the apoptotic subpopu-lation to about �10% of the entire population. In contrast, EGF andPDGF showed only marginal effects on viability. Rapamycin treat-ment resulted in �80% of cells undergoing apoptosis. Cells exposedto rapamycin appeared to lose membrane integrity late in apoptosisyet remained attached to the culture dish. Addition of IGF-I or insulinresulted in essentially complete protection from apoptosis, whereasEGF increased the proportion of viable rapamycin-treated cells from21% to 50%; PDGF increased the proportion to 58%. To demonstraterapamycin treatment inhibited mTOR activity under these growthconditions the phosphorylation status of a downstream substrate,

p70S6K, was examined after treatment with rapamycin or inhibitorsof PI3K. As shown in Fig. 1, 2-h pretreatment with rapamycin (100ng/ml) or the PI3K inhibitors, wortmannin (0.93 �M) and LY294002(20 �M), completely blocked IGF-I-stimulated activation of p70S6K.Consistent with this data, we have shown previously that IGF-I doesnot reactivate mTOR in rapamycin-treated cells (42).

PD 98059 Inhibits IGF-I-stimulated Erk1 and Erk2 Activity inRh1 Cells. Because the Ras-Raf-MEK-MAP kinase pathway medi-ates many of the known effects of IGF-I on cell proliferation, we used

Fig. 2. PD 98059 inhibits IGF-I stimulation of Erk1 and Erk2 phosphorylation for upto 6 days. A, serum-starved Rh1 cells were treated with PD 98059 (10–10,000 nM) for 2 hbefore they were stimulated with IGF-I. The cell lysates were subjected to Western blotanalysis with an antibody specific for phsopho-Erk1 and phospho-Erk2. B, Rh1 cellsgrown in MN2E medium were exposed to 15 �M PD 98059 and incubated for differentperiods of time. At each time point, the cells were stimulated with IGF-I (10 ng/ml) for5 min, and phosphorylation of Erk1 and Erk2 was determined as above. C, Rh1 cells weregrown in MN2E medium for 1 or 6 days in the presence of 15 �M PD 98059. Phospho-Erk1 and phospho-Erk2 were detected after stimulation with IGF-I for 5 min. The resultsare representative of those of four independent experiments.

Fig. 3. Expression of dominant-negative RasN17 completely suppresses Ras activationin Rh1 cells. A, Rh1 or Rh1 cells infected with MSCV-I-GFP/RasN17 or controlMSCV-I-GFP virus were serum-starved for 36 h, stimulated with IGF-I (10 ng/ml) for 5min, and then lysed. Cleared lysates were incubated with glutathione-agarose beads boundto a GST-Raf1 RBD fusion protein (GST-RBD). The beads were then washed, and theproteins were resolved by SDS-PAGE. The amount of activated Ras bound to theGST-RBD beads was determined by anti-Ras immunoblotting (top panel). Cell lysateswere also directly subjected to anti-Ras immunoblotting to determine levels of Ras in eachsample (bottom panel). The blots are representative of experiments that were replicatedthree or four times. B, Rh1 cells or Rh1 cells infected with MSCV-I-GFP or MSCV-I-GFP/RasN17 were grown in MN2E medium for 24 h then incubated an additional 2 h withor without PD 98059 (15 �M). Subsequently they were stimulated with EGF. Phospho-Erk1 and phospho-Erk2 were detected after 5 min of stimulation with EGF (25 ng/ml; toppanel) by Western blot using an anti-phospho-Erk1 and phospho-Erk2 antibody. Themembranes were stripped and incubated with an antibody that recognized total Erk1 andErk2 (bottom panel). The results that are shown are representative of those of twoindependent experiments. C, experimental details are as described for B, but cells werestimulated with IGF-I (10 ng/ml). The results of analysis with the anti-phospho-Erk1 andphospho-Erk2 antibody are shown in the top panel; the results of analysis with theanti-Erk1 and Erk2 antibody are in the center panel. Phosphorylation of Akt (Ser473) wasdetected after 5 min of stimulation with IGF-I. The membranes were stripped andincubated with anti-Akt antibody to ensure that equal amounts of protein were loaded ineach lane. These results are representative of those of two independent experiments.

Table 2 IGF-I prevents rapamycin-induced apoptosis of PD 98059-treated Rh1 cellsa

Treatmentb

Percentage of cellsa

Viable Apoptoticc

Control 70 30IGF-I 83 17PD 98059 57 43PD 98059 � IGF-I 80 20Rapamycin 23d 89Rapamycin � IGF-I 84 18Rapamycin � PD 98059 21d 80Rapamycin � PD 98059 � IGF-I 76 25

a Results show mean values from two independent experiments (means differed by�6%).

b IGF-I, 10 ng/ml; PD 98059, 15 �M; rapamycin, 100 ng/ml.c Necrosis, Annexin V-negative, propidium iodide-positive: �0.6%.d P � 0.05.

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a specific inhibitor of MEK1, PD 98059 (43, 44), to assess the role ofMEK1 in the antiapoptotic signaling pathways induced by IGF-I. Rh1cells were serum-starved for 36 h and exposed for 2 h to PD 98059 (10nM to 15,000 nM) before stimulation with IGF-I (10 ng/ml). Westernblot analysis detected phosphorylated Erk1 and Erk2 in lysates of cellsthat had been stimulated with IGF-I for 5 min (the time of maximalstimulation). PD 98059 inhibited Erk1 and Erk2 phosphorylation in aconcentration-dependent fashion (Fig. 2A). IGF-I signaling was com-pletely inhibited by 3 �M of PD 98059. To determine how quickly PD98059 inhibited IGF-I activation of Erk1 and Erk2, we exposed cellsgrown in MN2E medium to 15 �M of PD 98059 and incubated thecells 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 anincubation time of 30 min was sufficient to completely abrogate theIGF-I-induced phosphorylation of Erk1 and Erk2. To determinewhether this phosphorylation was inhibited for prolonged periods,Rh1 cells were grown for 1 or 6 days in the presence of PD 98059 (15�M) and then subjected to IGF-I treatment (10 ng/ml) for 5 min.IGF-I-stimulated phosphorylation of the two proteins in cells grown inPD 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-dependentmanner.

IGF-I Protected PD 98059-treated Cells from Rapamycin-induced Apoptosis. Having established conditions for prolonged in-hibition of MEK1 signaling by PD98059, we determined whetherIGF-I could still prevent rapamycin-induced apoptosis. Rh1 cellsgrown in MN2E medium were exposed to 0.1% DMSO (vehiclecontrol), 15 �M of PD 98059, 100 ng/ml rapamycin, or 15 �M ofPD98059 and 100 ng/ml rapamycin for 4 days with or withoutcoincubation with IGF-I (10 ng/ml). Apoptosis was quantitated byusing the ApoAlert flow cytometric assay. Approximately 30% ofcells in the control population were annexin V-positive and propidiumiodide-positive in the absence of IGF-I; however, the fraction de-creased to �17% when cells were treated with IGF-I (Table 2). PD98059 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 rapa-mycin induced a significant increase in the proportion of apoptoticcells (�78% apoptosis). IGF-I completely abrogated apoptosis ofcells treated with rapamycin alone or almost completely with rapa-mycin plus PD98059 (76% viability). Therefore, under conditions inwhich PD98059 inhibited MEK1 signaling, IGF-I protected cells fromrapamycin-induced apoptosis. These results suggest that activation ofErk1 and Erk2 is not required for IGF-I-mediated protection fromrapamycin-induced apoptosis.

Retroviral Infection of Rh1 Cells with GFP-tagged Dominant-Negative RasN17. Our results with pharmacologic inhibitors dem-onstrated that IGF-I-mediated protection of Rh1 cells from rapamy-cin-induced apoptosis is independent of the Ras-Erk1-Erk2 signalingpathway. To independently confirm this result, we performed exper-iments using a genetic approach in which an allele that encoded adominant-negative form of Ras (RasN17) was overexpressed. Rh1cells were infected with MSCV-I-GFP/RasN17 or control MSCV-I-GFP virus. The level of expression of RasN17 greatly exceededendogenous Ras (�100-fold) for up to 5 days after infection. Further-more, IGF-I did not activate Ras 5 days after infection (data notshown). Infected cells and uninfected control cells were serum-starvedfor 36 h and stimulated with IGF-I (10 ng/ml) for 5 min. The amountof activated Ras was measured using a commercially available Rasactivation kit, in which only activated Ras (Ras-GTP) binds to the

Fig. 4. Effect of growth factors on the phosphorylation of Akt in Rh1 cells. A, Rh1 cellswere serum-starved for 36 h and stimulated with IGF-I (10 ng/ml; top panel), EGF (25ng/ml; center panel) or PDGF (25 ng/ml; bottom panel) for up to 120 min. The results ofWestern blot analysis of phospho-Akt (Ser473) and Akt (phosphorylated and nonphos-phorylated) are shown. Similar results were obtained in four independent experiments. B,phosphorylation of Akt induced by IGF-I, EGF, or PDGF is mediated by the PI3Ksignaling pathway in Rh1 cells. Cells were serum-starved for 36 h and exposed towortmannin (0.3–2.0 �M) or LY 294002 (20 �M or 40 �M) for 2 h before stimulation withIGF-I (10 ng/ml; top panel), EGF (25 ng/ml; center panel) or PDGF (25 ng/ml; bottompanel). Akt phosphorylation was detected after 5 min of stimulation. The same mem-branes were stripped of bound antibody and incubated with an anti-Akt antibody. Totallevels of Akt protein are shown in the bottom panels for the respective growth factors. Theresults were similar in at least three independent experiments.

Table 3 IGF-I inhibits rapamycin-induced apoptosis of Rh1 cells expressing RasN17a

Cell line � treatmentb

Percentage of cells SDa

Viable Apoptoticc

Rh1 83.1 0.2 15.4 0.4Rh1 � IGF-I 89.1 1.1 10.9 1.3Rh1/GFP/RasN17 84.4 4.1 15.2 2.1Rh1/GFP/RasN17 � IGF-I 93.2 0.6 6.6 0.2Rh1/GFP/RasN17 � rapamycin 60.2 6.6 38.7 4.0Rh1/GFP/RasN17 � rapamycin � IGF-I 91.1 0.7 8.7 0.5

a Results are mean SD (n 3).b Rh1/GFP/RasN17, Rh1 cells infected with MSCV-I-GFP/RasN17; Treatment, IGF-I,

10 ng/ml; rapamycin, 100 ng/ml.c Annexin V-negative, propidium iodide-positive: �1.5%.

Table 4 IGF-I protects Rh1 cells from rapamycin-induced apoptosis in the presence ofLY 294002a

Treatmentb

Percentage of cells SDa

Viable Apoptoticc

Control 77.9 1.0 22.1 0.9IGF-I 84.4 0.3 15.5 0.2LY 294002 55.9 8.7 44.1 4.3LY 294002 � IGF-I 83.2 1.0 16.7 1.0LY 294002 � Rapamycin 32.7 1.4d 66.9 0.9LY 294002 � Rapamycin � IGF-I 81.2 2.5 18.7 1.3

a Results are mean SD (n 3).b IGF-I, 10 ng/ml; Rapamycin, 100 ng/ml; LY 294002, 20 �M.c Necrosis: Annexin V-negative, propidium iodide-positive: �0.4%.d P � 0.05.

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RBD of Raf1. Active GTP-bound Ras was precipitated from celllysates with the GST-Raf1 RBD coupled to glutathione-agarose, andthe fraction of activated Ras was evaluated by immunoblotting usingan anti-Ras antibody. Ras coimmunoprecipitated with GST-Raf1RBD from IGF-I-treated uninfected Rh1 cells or Rh1 cells infectedwith MSCV-I-GFP, but activated Ras was not detected in IGF-I-treated Rh1 cells infected with the virus expressing RasN17 (Fig. 3A).These results suggest that Ras activation was completely suppressedby the dominant-negative RasN17. Western blot analysis using thesame anti-Ras antibody as was used in the activated Ras assaysshowed that cells expressing dominant-negative RasN17 have higherlevels of Ras than Rh1 cells that are uninfected or are infected with thecontrol vector.

To determine whether RasN17 inhibits cellular Ras signaling, weanalyzed the phosphorylation status of Erk1 and Erk2 in Rh1 cellsalone, and in Rh1 cells that were infected with MSCV-I-GFP orMSCV-I-GFP/RasN17 and stimulated with EGF (25 ng/ml) or IGF-I(10 ng/ml) for 5 min. Overexpression of RasN17 clearly inhibitedErk1 and Erk2 phosphorylation induced by EGF or IGF-I (Fig. 3, Band C, top panel). Thus, results obtained with this dominant-negativeRas are in contrast to the results obtained with the pharmacologicalinhibitor of farnesyltransferase (above). PD 98059 treatment (15 �M),which was used as a positive control, resulted in the complete block-ade of the ability of the growth factor to activate Erk1 or Erk2 in Rh1cells alone and in Rh1 cells infected with MSCV-I-GFP. Unlike itsinhibition of IGF-I-mediated activation of Erk1 and Erk2, RasN17 did

not inhibit IGF-I-mediated activation of Akt (Fig. 3C). These findingsindicate that Akt is not a downstream target of Ras in the IGF-Isignaling pathway of Rh1 cells.

IGF-I Inhibits Rapamycin-induced Apoptosis of Rh1 Cells ThatOverexpress RasN17. Uninfected Rh1 cells and those stably ex-pressing MSCV-I-GFP/RasN17 were preincubated with or withoutrapamycin (100 ng/ml) for 4 days. Duplicate samples were coincu-bated with or without IGF-I (10 ng/ml). The percentage of apoptoticcells within each population was then evaluated. In populations ofRh1 cells and those expressing GFP/RasN17, both of which weregrown in MN2E medium, �17% of the cells were apoptotic (Table 3).Rapamycin treatment for 4 days substantially increased the proportionof apoptotic cells in the populations expressing GFP/RasN17 (�40%).IGF-I treatment of cell populations expressing GFP/RasN17 de-creased the percentage of apoptotic cells to their respective controllevels. The level of necrotic cells was similar (�2%) in both popu-lations of Rh1 cells and those expressing GFP/RasN17 cells underthese conditions. Results for Rh1 cells expressing only GFP weresimilar to parental Rh1 cells (data not shown). These results clearlysuggest that suppression of apoptosis by IGF-I is not Ras-dependent.

Effect of Growth Factors on the Phosphorylation of Akt in Rh1Cells. Many growth factors reportedly exert antiapoptotic effects invarious cell types by activating PI3K and Akt (protein kinase B; Ref.45). Because IGF-I, EGF, and PDGF stimulate the PI3K-Akt signal-ing pathway, we tested the role of Akt signaling in IGF-I-mediatedprotection from rapamycin-induced apoptosis. After 36 h of serumstarvation, Rh1 cells were stimulated with IGF-I (10 ng/ml), EGF (25ng/ml), or PDGF (25 ng/ml) for up to 120 min. Phosphorylation ofAkt at Ser473 served as the indicator of activation. Serum starvationresulted in a low level of detectable phospho-Ser473 (Fig. 4A). Aktactivation was evident after 5 min of treatment with any of the threegrowth factors. Stimulation by EGF was transient and returned to thebasal levels within 30 min. In contrast, phosphorylation of Akt byPDGF was more prolonged, and Akt phosphorylation by IGF-I re-mained stable for at least 2 h. The total amount of Akt was unchangedafter growth factor stimulation. These results indicate that IGF-I,EGF, and PDGF receptors mediate activation of the Akt pathway inRh1 cells.

Phosphorylation of Akt by IGF-I, EGF, and PDGF Is Mediatedby the PI3K Pathway. To determine whether the PI3K signalingpathway is responsible for the Akt activation stimulated by IGF-I,EGF, or PDGF, we used the PI3K inhibitor LY 294002 to block PI3Kactivation. Serum-starved Rh1 cells were exposed to LY 294002 (20�M or 40 �M) for 2 h before they were stimulated with IGF-I, EGF,or PDGF. Akt phosphorylation was detected after 5 min of stimula-tion, as described in the preceding section. LY 294002 (40 �M)effectively blocked growth factor-induced phosphorylation of Akt(Fig. 4B).

IGF-I Prevents Rapamycin-induced Apoptosis of Rh1 CellsTreated with LY 294002. To determine whether the effect of IGF-Ion inhibition of rapamycin-induced apoptosis involved the PI3Kpathway, we treated Rh1 cells grown in MN2E medium with DMSO(0.1%), rapamycin (100 ng/ml), LY 294002 (20 �M), or rapamycin(100 ng/ml) and LY 294002 (20 �M). Duplicate samples were coin-cubated with or without IGF-I (10 ng/ml). Our assessment of apo-ptosis indicated that 23% of the untreated cell populations consisted ofannexin V-positive and propidium iodide-positive cells, but IGF-Ireduced this proportion to 15% (Table 4). LY 294002 treatmentresulted in apoptosis of �54% of cells. When the cells were exposedto LY 294002 and rapamycin, only 33% of cells were scored asviable. Coincubation with IGF-I almost completely protected cellsunder all conditions of drug exposure. Thus, concentrations of LY294002 that markedly inhibit PI3K activity failed to blunt the anti-

Fig. 5. Overexpression and function of a dominant-negative Akt mutant in Rh1 cells.A, Rh1 cells were stably transfected with pUSE vector alone or with pUSE encoding ac-Myc-tagged dominant-negative form of Akt (pUSE-dnAkt). Cells grown in MN2Emedium were stimulated with IGF-I for 10 min. The cell lysates subjected to immuno-precipitation with anti-c-MYC antibody. The precipitated proteins were resolved bySDS-PAGE and immunoblotted using an anti-Akt antibody. The results that are shown arerepresentative of five experiments. B, activation of Akt in cells described for A wasdetermined by Western blot analysis using the anti-phospho-Akt (Ser473) antibody (toppanel). The center panel shows the results of Western blot analysis with the anti-Aktantibody to confirm that equal amounts of protein were loaded. Bottom panel shows theresults of the in vitro kinase assay using the anti-phospho-GSK-3 antibody. The resultsshown are representative of those of three independent experiments.

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apoptotic effect of IGF-I. Moreover, IGF-I prevented apoptosis in-duced by these agents either alone or in combination with rapamycin.IGF-I also partially rescued from higher concentrations of LY294002(50 and 100 �M) that completely abrogate PI3K signaling. Thus, at100 �M of LY294002 IGF-I increased the proportion of viable cellsfrom 19% to 59%. Qualitatively similar results were obtained usingthe structurally distinct PI3K inhibitor wortmannin (data not shown).Because Akt is an effector of survival signaling and is downstream ofPI3K, our results can be explained in two ways: (a) Akt may beactivated in a PI3K-independent fashion; or (b) an Akt-independentpathway may be used. If the first explanation were correct Aktactivation induced by IGF-I would not be inhibited by LY 294002.However, IGF-I-mediated activation of endogenously expressed Aktwas sensitive to PI3K inhibitors, a result that favors the existence ofan Akt-independent pathway in Rh1 cells.

Overexpression of a Dominant-negative Akt Mutant in Rh1Cells. To independently determine whether Akt activation is requiredfor IGF-I-induced survival signaling, we examined the consequencesof overexpressing a kinase-deficient dominant-negative Akt in Rh1cells. We stably transfected the cells with the pUSE control vector orthe pUSE vector encoding a c-Myc epitope-tagged dominant-negativeAkt mutant that lacked kinase activity (pUSE-dnAkt). After G418selection-resistant clones were screened for expression of c-Myc-tagged Akt (Fig. 5A), the anti-c-Myc antibody was used to immuno-precipitate the tagged protein, which was then subjected to immuno-blotting analysis with an anti-Akt antibody. The Akt band wasdetected in cells transfected with the dominant-negative Akt expres-sion plasmid but not in parental Rh1 cells or in cells transfected withcontrol 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) for10 min, and then lysed the cells on ice. Activation of Akt by IGF-Iwas evaluated by assessing the phosphorylation of Ser473 of Akt orthe in vitro kinase activity of protein immunoprecipitated by theanti-Akt antibody. IGF-I stimulated phosphorylation of Ser473 inparental Rh1 cells and Rh1 cells transfected with pUSE, but not inthose transfected with pUSE/dnAkt (Fig. 5B, top panel). After themembrane was stripped of bound antibody, the membrane was incu-bated with the anti-Akt antibody to determine the total amount of Aktin each cell line (Fig. 5B, center panel). The total amount of Akt was4-fold greater in Rh1 cells transfected with pUSE/dnAkt than inparental or vector-transfected cells.

To determine whether changes in Akt phosphorylation were corre-lated with alterations in kinase activity, we examined the phospho-rylation status of a target downstream from Akt, e.g., GSK-3. Cellswere stimulated with IGF-I, and Akt (phosphorylated and unphospho-rylated) was immunoprecipitated. Immunoprecipitates were used invitro to phosphorylate a GSK-3 fusion protein. Phosphorylation wasreduced in cells transfected with the dominant-negative Akt expres-sion plasmid but not in parental Rh1 cells or Rh1 cells transfectedwith control vector (Fig. 5B, bottom panel). These results indicate thatthe dominant-negative Akt effectively blocks the activity of endoge-nous Akt.

IGF-I-mediated Protection of Rh1 Cells From Rapamycin-induced Apoptosis Is Independent of Akt Signaling. To determinewhether IGF-I-mediated inhibition of rapamycin-induced apoptosis isindependent of Akt signaling, we exposed cells expressing dominant-negative Akt to rapamycin (100 ng/ml) for 4 days in the presence orabsence of IGF-I (10 ng/ml), and then determined the extent of

Fig. 6. IGF-I-mediated protection of Rh1 cells fromrapamycin-induced apoptosis is independent of the PI3K-Akt signaling pathway. Rh1 cells (A), Rh1 cells transfectedwith pUSE (B), and Rh1 cells transfected with pUSE-dnAkt (C) were grown in MN2E medium and treated with orwithout rapamycin (100 ng/ml) for 4 days. During thattime, duplicate samples were with or without IGF-I (10ng/ml). Apoptosis was evaluated by using the ApoAlertassay. The percentage of distribution of cells in each quad-rant is presented. Similar results were obtained in fourindependent experiments. In the top panels, the ordinate isthe uptake of propidium iodide (PI); the abscissa, annexinV-FITC fluorescence. Viable cells are represented in thebottom left quadrant. The bottom panels show the corre-sponding distribution of annexin V-FITC staining of cellpopulations.

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apoptosis within each treatment population. The level of viability wasslightly lower in this experiment: 35% of cells within the controlpopulation were both annexin V- and propidium iodide-positive (Fig.6). IGF-I reduced this level to 14–19%. When the cells transfectedwith either pUSE-dnAkt or pUSE were treated with rapamycin, theproportion 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 sug-gests that the protection is independent of PI3K and Akt activity.

IGF-I Prevents Rapamycin-induced Apoptosis of Rh1 CellsExpressing Dominant-Negative RasN17 in the Presence of Inhib-itors of MEK1 and PI3K. We determined whether IGF-I couldprotect Rh1 cells from apoptosis after two major survival-signalingpathways were blocked by simultaneous genetic and pharmacologicapproaches. Rh1 cells expressing MSCV-I-GFP/RasN17 were incu-bated with DMSO (0.1%), rapamycin (100 ng/ml), PD 98059 (15 �M),or LY 294002 (20 �M) for 40 h. In addition, other samples of cellswere treated with rapamycin and each of the inhibitors. Duplicatesamples were coincubated with IGF-I (10 ng/ml). The extent ofapoptosis was assessed. Analysis of the results revealed that �10% ofuntreated cells were apoptotic (Fig. 7). Approximately 20% of the

Fig. 7. IGF-I protects Rh1 cells against rapamycin-induced apoptosis independent of both Ras-Erk1-Erk2and PI3K-Akt activity. Rh1 cells infected with MSCV-I-GFP/RasN17 were grown under serum-free conditionsand treated with DMSO 0.1% (control), rapamycin (100ng/ml), IGF-I (10 ng/ml) or both rapamycin plus IGF-I(top data set). Center data set (LY294002): cells weretreated as above, but in addition each sample containedLY294002 (20 �M). Lower data set (PD98059): cellswere treated as in the top data set, but in additionPD98059 (15 �M) was added to each sample. In the toppanels, the ordinate is the uptake of propidium iodide(PI); the abscissa, annexin V-FITC fluorescence. Viablecells are represented in the bottom left quadrant. Thebottom panels show the corresponding distribution ofannexin V-FITC staining of cell populations. Apoptosiswas determined after 40 h by quantitative FACS analysis(ApoAlert). The percentage of distribution of cells ineach quadrant is presented. Similar results were ob-tained in at least four independent experiments.

Table 5 IGF-I rescues Rh1 cells transfected with pUSE-dnAkt from rapamycin-inducedapoptosis in the presence of LY 294002 or PD 98059a

Treatmentb

Percentage of cells SDa

Viable Apoptoticc

Control 74.1 4.9 25.1 2.6IGF-I 81.2 2.6 17.2 1.3LY 294002 42.8 3.7d 56.5 5.9LY 294002 � IGF-I 79.5 3.3 19.8 2.2LY 294002 � rapamycin 31.4 0.7d 67.9 5.5LY 294002 � rapamycin � IGF-I 77.9 4.1 22.0 2.0PD 98059 77.9 5.5 21.8 2.7PD 98059 � IGF-I 85.6 4.5 14.2 2.1PD 98059 � rapamycin 64.6 3.1 34.7 2.1PD 98059 � rapamycin � IGF-I 82.4 5.8 17.3 3.0

a Results are mean SD (n 3).b IGF-I, 10 ng/ml; Rapamycin, 100 ng/ml; LY 294002, 20 �M; PD 98059, 15 �M.c Necrosis: Annexin V-negative, propidium iodide-positive: �0.9%.d P � 0.05.

Table 6 IGF-I rescues Rh30 cells from rapamycin-induced apoptosis in the presenceof LY 294002a

Treatmentb

Percentage of cells

Viable Apoptoticc

Control 78 23IGF-Ib 88 13LY 294002 20d 78LY 294002 � IGF-I 68 33LY 294002 � rapamycin 18d 82LY 294002 � rapamycin � IGF-I 58 42

a This table shows mean results from two independent experiments (means differed by�7%).

b IGF-I, 10 ng/ml; Rapamycin, 100 ng/ml; LY 294002, 30 �M.c Necrosis, Annexin V-negative, propidium iodide-positive: �1%.d P � 0.05.

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population treated with only rapamycin were apoptotic. In contrast,treatment with LY 294002 alone resulted in �53% apoptosis; and PD98059, 13%. Treatment with rapamycin and each of the other signal-ing inhibitors resulted in essentially additive levels of apoptosis. IGF-I(10 ng/ml) completely inhibited apoptosis induced by rapamycin inthe presence or absence of the other signaling inhibitors (Fig. 7).Taken together these results additionally support our findings thatIGF-I prevents rapamycin-induced apoptosis of Rh1 cells in a Ras-MEK-Erk1-Erk2/PI3K-independent manner.

IGF-I Protects Rh1 Cells From Rapamycin-induced ApoptosisDespite Simultaneous Inhibition of PI3K-Akt and MAPK Path-ways. The reciprocal experiment in which Akt function was geneti-cally abrogated was next performed. Rh1 cells stably expressingdnAkt were exposed to LY 294002 (20 �M) or PD 98059 (15 �M) for24 h. In addition, other samples of cells were treated with rapamycinand each of the inhibitors. Duplicate sets of samples were exposedwith or without IGF-I (10 ng/ml). The flow cytometric assay showedthat �25% of untreated cells were apoptotic (Table 5). Treatment withLY 294002 or PD 98059 resulted in apoptosis of 58% and 22% ofcells, respectively. However, LY 294002 or PD 98059 in combinationwith rapamycin increased the proportion of apoptotic cells to 69% and35%, respectively. Important is the finding that IGF-I preventedapoptosis caused by these agents either separately or in combination.

IGF-I Protects Rh30 Cells From Rapamycin-induced ApoptosisDespite Simultaneous Inhibition of PI3K-Akt and MAPK Path-ways. Results presented demonstrate protection from rapamycin-induced apoptosis in the presence of pharmacological inhibitors ofPI3K/Akt or Ras/Erk1/2 pathways in Rh1 cells. We next extended thestudy to Rh30 rhabdomyosarcoma cells, as we have reported previ-ously that IGF-I protects against rapamycin-induced apoptosis in thiscell line (35). Because of the instability of LY294002 under cultureconditions Rh30 cells were treated with LY294002 and rapamycinwith or without IGF-I for 45 h, and the level of apoptosis determined(Table 6). IGF-I partially rescued cells from apoptosis induced byLY294002 alone or in combination with rapamycin. IGF-I also res-cued cells from apoptosis induced by PD98059 as a single agent, orwhen combined with rapamycin and exposed for 6 days (Table 7).Furthermore, IGF-I prevented apoptosis induced when Rh30 cellswere exposed to PD98059 combined with LY98059 for 45 h (Fig. 8).The combination of PD98059 plus LY294002 resulted in �78% lossof viability, whereas in the presence of IGF-I this was reduced to�33%.

DISCUSSION

The primary aim of the present study was to determine whether theRas-Erk1/2 and PI3K-Akt pathways are required for growth factor-mediated prevention of rapamycin-induced apoptosis of Rh1 cells.

Both pathways are implicated in survival signaling in various cancercells, and are activated downstream of the growth factor receptors forIGF-I, insulin, EGF, and PDGF. In agreement with previous findings,our results showed that IGF-I completely rescued Rh1 and Rh30 cellsfrom cytotoxic effects of rapamycin (42). Insulin also preventedrapamycin-induced apoptosis in Rh1 and Rh30 (data not shown), butneither PDGF nor EGF were particularly effective. Clearly, theseresults suggest that the Rh1 cell line displays distinct patterns ofgrowth factor-mediated survival and that IGF-I is the growth factormost effective in preventing rapamycin-induced apoptosis. Therefore,

Fig. 8. IGF-I partially protects Rh30 cells from apoptosis despite simultaneous inhi-bition of PI3K-Akt and MAP kinase pathways. Rh30 cells were grown under serum-freeconditions and treated with inhibitors of MEK1 and PI3K with or without IGF-I (10ng/ml) for 45 h. A, DMSO 0.1% (control), IGF-I (10 ng/ml). B, PD98059 (15 �M). C, LY294002 (20 �M). D, PD98059 (15 �M) plus LY 294002 (20 �M). Ordinate is the uptakeof propidium iodide (PI); the abscissa, annexin V-FITC fluorescence (top panels). Viablecells are represented in the bottom left quadrant. The bottom panels show the correspond-ing distribution of annexin V-FITC staining of cell populations. Apoptosis was determinedafter 45 h by quantitative FACS analysis (ApoAlert). The percentage of distribution ofcells in each quadrant is presented.

Table 7 IGF-I rescues Rh30 cells from rapamycin-induced apoptosis in the presenceof PD 98059a

Treatmentb

Percentage of cellsa

Viable Apoptoticc

Control 72 29IGF-I 93 8PD 98059 42 58PD 98059 � IGF-I 89 10PD 98059 � rapamycin 13d 88PD 98059 � rapamycin � IGF-I 68 34

a This table shows mean results from two independent experiments (means differed by�5%).

b IGF-I, 10 ng/ml; Rapamycin, 100 ng/ml; PD 98059, 15 �M.c Necrosis, Annexin V-negative, propidium iodide-positive: �1%.d P � 0.05.

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the remainder of our experiments focused on understanding the mech-anism whereby IGF-I prevents rapamycin-mediated apoptosis of thesecell lines. In particular, we additionally investigated whether the twosignaling pathways activated by IGF-I, Ras-Raf-MEK-Erk1-Erk2(MAPK pathway), and PI3K-Akt are involved in mediating protectionfrom rapamycin-induced apoptosis.

The MAPK pathway is responsible for mediating numerous effectsof IGF-I (46, 47) in part through a Ras-dependent activation ofRaf/MEK1 leading to activation of Erk1 and Erk2. Prolonged inhibi-tion of Erk1/2 activation by PD 98059, a specific noncompetitiveinhibitor of MEK1, with respect to ATP binding (43), in the presenceof rapamycin induced �75% apoptosis that was almost completelyabrogated by IGF-I. Thus, our results demonstrate that MEK1 acti-vation of Erk1/2 MAPK pathway is not essential for IGF-I protectionfrom rapamycin-induced apoptosis. Similar results were obtained inRh30 cells.

We next examined whether Ras signaling was necessary for IGF-I-mediated protection. Ras signaling was abrogated genetically byinfecting Rh1 cells with a retrovirus carrying a dominant-negativemutant of Ras (RasN17). The dominant-negative RasN17 was ex-pressed at high levels for at least 5 days after infection of Rh1 cellsand inhibited Ras activation by IGF-I. RasN17 suppressed Erk-1/2phosphorylation completely, suggesting that SCH66336 incompletelyinhibits this pathway in Rh1 cells. Importantly, IGF-I completelyprotected the RasN17 expressing cells from rapamycin-induced ap-optosis. Taken together, these data appear to confirm that Ras signal-ing is not required for IGF-I to protect against rapamycin-inducedapoptosis. Our data also indicate that Ras is not upstream of Akt, asphosphorylation of Akt by IGF-I was not inhibited in RasN17-expressing cells. Hence, these results differ from reports implicatingRas/Akt signaling in survival (48).

We next focused on the role of PI3K-mediated activation of Akt insuppression of apoptosis (49, 50). The protective effect of IGF-I mayresult 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 foractivation (53, 54). Because PI3K is known to activate Akt we firstdetermined the effect of IGF-I, EGF, and PDGF on Akt activation.IGF-I treatment of cells resulted in a sustained phosphorylation ofAkt, whereas after stimulation by EGF phosphorylation declinedwithin 30 min to basal level. The effect of PDGF was intermediate.Thus, there was some correlation with the ability of these growthfactors to protect cells from rapamycin-induced apoptosis. As antici-pated, activation of Akt by IGF-I, EGF, or PDGF was inhibited by LY294002. The ability of this PI3K inhibitor to decrease Akt phospho-rylation suggests that the activation of Akt is PI3K-dependent, al-though we have not directly assayed PI3K activity in Rh1 cells. Inreported studies (55, 56) IGF-I protection of cells from apoptosisrequired activation of PI3K, and activation of Akt was sufficient forprotection (32).

Because of the potential breakdown of LY294002, assays for ap-optosis were limited to 24–45 h exposures. LY 294002 with orwithout rapamycin for 24 h increased the proportion of apoptotic cellsto �54 and 67%, respectively, but addition of IGF-I could completelyovercome this effect and protect the cells from death. Thus, theconcentration of LY 294002 that produced very marked inhibition ofPI3K signaling was unable to attenuate the antiapoptotic effect ofIGF-I. Apoptosis at higher concentrations of LY294002 that com-pletely inhibit PI3K signaling was also significantly inhibited byIGF-I. However, apoptosis caused by such drug concentrations maybe in part independent of the inhibition of PI3K. However, our resultsindicate that rescue by IGF-I from rapamycin-induced apoptosis ispredominantly independent of the PI3K/Akt signaling pathway.

To additionally test whether PI3K/Akt pathway was required forIGF-I-induced cell survival, we used a genetic approach. Expressionof the dominant-negative Akt significantly inhibited phosphorylationof Akt (Ser473) and endogenous Akt kinase activity in response toIGF-I stimulation, thus demonstrating function. However, massiveapoptosis (�75%) induced by rapamycin was completely protected byIGF-I in Rh1/dnAkt cells. Thus, Akt activity is not required forsurvival mediated by IGF-I. The PI3K inhibitor also caused a highlevel of apoptosis (�60%) in cells expressing dnAkt. This suggeststhat Akt is not the major downstream target of PI3K involved in cellsurvival in these cells.

There is accumulating evidence indicating various levels of cross-talk between the MAPK and the PI3K/Akt pathways, which may playa role in regulating apoptosis. Thus, it is possible that Ras andPI3K/Akt pathways could be redundant, and that blocking each indi-vidually still allows IGF-I-mediated protection from apoptosis. There-fore, we have investigated whether IGF-I could still protect cells fromapoptosis after simultaneously blocking these two major signalingpathways by genetic and pharmacological approaches. IGF-I pro-tected against rapamycin-induced apoptosis in Rh1 cells expressingdominant-negative RasN17 in the presence of PI3K inhibitor, MEK1inhibitor, or both agents. Similarly, IGF-I completely protected Rh1cells expressing dominant-negative Akt exposed to rapamycin, LY294002, and PD 98059. Thus, simultaneous inhibition of both PI3K/Akt and Ras signaling pathways did not abrogate the protective actionof IGF-I. Similarly, IGF-I significantly protected Rh30 cells treatedwith a combination of LY294002 and PD98059. Taken together, thesedata additionally support the contention that neither the MAPK norPI3K/Akt-dependent pathways are required for IGF-I to protectagainst rapamycin-induced apoptosis.

Our studies have focused on determining whether signaling throughPI3K/Akt or Ras/Erk1/2 is involved in IGF-I-mediated protection ofrapamycin-induced apoptosis. Survival in Rh1 and Rh30 cells appearsindependent of either pathway. IGF-I can activate other MAPKs, suchas c-Jun NH2-terminal kinase or p38, and these pathways have notbeen studied. Consequently, additional studies are needed to identifythe signaling pathway(s) by which IGF-I protects Rh1 cells fromapoptosis. As mTOR inhibition by rapamycin mimics certain effectsof amino acid or glucose deprivation (57, 58), it is possible that theeffects of IGF-I or insulin on nutrient regulation may play a role inprotection from apoptosis. The identification and characterization ofthese novel pathways and the development of inhibitors of these novelpathways may be important for enhancing the cytotoxic effect ofagents that target mTOR, such as CCI-779 and the RAD001 rapamy-cin analogs that are currently in Phase I/II trials as cancer treatment.

ACKNOWLEDGMENTS

We thank Richard Ashmun for FACS analysis and Julia Cay Jones forassistance in editing this manuscript.

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2003;63:364-374. Cancer Res   Kuntebommanahalli N. Thimmaiah, John Easton, Shile Huang, et al.   Signaling Pathways

-Kinase-Akt′Ras-Erk1-Erk2 and Phosphatidylinositol 3Rapamycin-induced Apoptosis Is Independent of Insulin-like Growth Factor I-mediated Protection from

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