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Insulin-like Growth Factor 1 Inhibits Apoptosis Using the Phosphatidylinositol 3′-Kinase and Mitogen-activated Protein Kinase Pathways*

  • Marcelina Párrizas
    Footnotes
    Affiliations
    From the Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 and
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  • Alan R. Saltiel
    Affiliations
    Department of Physiology, University of Michigan, School of Medicine and the Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, Michigan 48105
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  • Derek LeRoith
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    From the Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 and
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    Recipient of Postdoctoral Fellowship EX-94 from the Ministerio de Educación y Ciencia (Spain).
Open AccessPublished:January 03, 1997DOI:https://doi.org/10.1074/jbc.272.1.154
      The role of insulin-like growth factor 1 (IGF-1) in preventing apoptosis was examined in differentiated PC12 cells. Induction of differentiation was achieved using nerve growth factor, and apoptosis was provoked by serum withdrawal. After 4-6 h of serum deprivation, apoptosis was initiated, concomitant with a 30% decrease in cell number and a 75% decrease in MTT activity. IGF-1 was capable of preventing apoptosis at concentrations as low as 10−9M and as early as 4 h. The phosphatidylinositol 3′ (PI3′)-kinase inhibitors wortmannin (at concentrations of 10−8M) and LY294002 (10−6M) blocked the effect of IGF-1. The pp70 S6 kinase (pp70S6K) inhibitor rapamycin (10−8M) was, however, less effective in blocking IGF-1 action. Moreover, stable transfection of a dominant-negative p85 (subunit of PI3′-kinase) construct in PC12 cells enhanced apoptosis provoked by serum deprivation. Interestingly, in the cells overexpressing the dominant-negative p85 protein, IGF-1 was still capable of inhibiting apoptosis, suggesting the existence of a second pathway involved in the IGF-1 effect. Blocking the mitogen-activated protein kinase pathway with the specific mitogen-activated protein kinase/extracellular-response kinase kinase inhibitor PD098059 (10−5M) inhibited the IGF-1 effect. When wortmannin and PD098059 were given together, the effect was synergistic. The results presented here suggest that IGF-1 is capable of preventing apoptosis by activation of multiple signal transduction pathways.

      INTRODUCTION

      Apoptosis or programmed cell death plays an important role in embryonic development, involution of organs, and tumorigenesis. During development of the nervous system, a large proportion of neurons die by this process; about 50% of embryonic postmitotic neurons ultimately die during the period when synapses are formed between neurons and their targets (
      • Oppenhein R.W.
      ,
      • Raff M.C.
      • Barres B.A.
      • Burne J.F.
      • Coles H.S.
      • Ishizaki Y.
      • Jacobson M.I.
      ). The survival of neurons is dependent on neurotrophins secreted by the target cells (
      • Batistatou A.
      • Greene L.A.
      • Cuello A.C.
      Neuronal Cell Death and Repair.
      ). In PC12 cells (rat pheochromocytoma cells) differentiated in the presence of nerve growth factor (NGF),
      The abbreviations used are: NGF
      nerve growth factor
      IGF-1
      insulin-like growth factor 1
      PI3′-kinase
      phosphatidylinositol 3′-kinase
      IRS-1
      insulin receptor substrate 1
      SH2
      Src homology region 2
      ERK
      extracellular-response kinase
      MAP
      mitogen-activated protein
      MAPK
      MAP kinase
      MEK
      MAPK/ERK kinase
      pp70S6K
      pp70 S6 kinase
      DMEM
      Dulbecco's modified Eagle's medium
      SFM
      serum-free DMEM
      PBS
      phosphate-buffered saline
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
      NGF itself, as well as other growth factors (including platelet-derived and epidermal growth factors, and insulin at high doses), protect cells maintained in serum-free media from apoptosis (
      • Yao R.
      • Cooper G.M.
      ).
      Insulin-like growth factor 1 (IGF-1) prevents apoptosis in a number of cell types. For example, IGF-1 at physiological concentrations was effective in inhibiting apoptosis in fibroblasts overexpressing c-myc (
      • Evan G.I.
      • Wyllie A.H.
      • Gilbert C.S.
      • Littlewood T.D.
      • Land H.
      • Brooks M.
      • Waters C.M.
      • Penn L.Z.
      • Hancock D.C.
      ,
      • Harrington E.A.
      • Bennett M.R.
      • Fanidi A.
      • Evan G.I.
      ), in cells treated with the topoisomerase II inhibitor etoposide (
      • Sell C.
      • Baserga R.
      • Rubin R.
      ), in neuroblastoma cells under hyperosmotic stress (
      • Matthews C.C.
      • Feldman E.L.
      ), and potassium-deprived cerebellar granule cells (
      • D'Mello S.R.
      • Galli C.
      • Ciotti T.
      • Calissano P.
      ,
      • Galli C.
      • Meucci O.
      • Scorziello A.
      • Werge T.M.
      • Calissano P.
      • Schettini G.
      ). Most cell types require IGF-1 for growth in culture (
      • Goldring M.B.
      • Goldring S.R.
      ), and a decrease in IGF-1 receptor number induces apoptosis in tumor cells (
      • Resnicoff M.
      • Abraham D.
      • Yutanawiboonchai W.
      • Rotman H.L.
      • Kajstura J.
      • Rubin R.
      • Zoltick P.
      • Baserga R.
      ). IGF-1 can also act as a survival factor in the absence of other factors. Specifically, IGF-1 inhibits apoptosis of several interleukin-3-dependent cell lines when IL-3 is removed (
      • Rodriguez-Tarduchy G.
      • Collins M.K.L.
      • Garcia I.
      • Lopez-Rivas A.
      ).
      Insulin and the IGFs exert their biological effects by binding to their respective transmembrane receptors. Insulin and IGF-1 receptors are similar, heterotetrameric proteins with intrinsic tyrosine kinase activity (
      • Ullrich A.
      • Bell J.R.
      • Chen E.Y.
      • Herrera R.
      • Petruzzelli L.M.
      • Dull T.J.
      • Gray A.
      • Coussens L.
      • Iiay Y.C.
      • Tsubokawa M.
      • Mason A.
      • Seeburg P.H.
      • Grunfeld C.
      • Rosen O.M.
      • Ramachandran J.
      ,
      • Ullrich A.
      • Gray A.
      • Tam A.W.
      • Yang-Feng T.
      • Tsubokawa M.
      • Collins C.
      • Henzel W.
      • LeBon T.
      • Kathuria S.
      • Chen E.
      • Jacobs S.
      • Francke U.
      • Ramachandran J.
      • Fujita-Yamaguchi Y.
      ). Both receptors are capable of binding insulin and IGF-1, but each receptor binds its own ligand with a 100-1000-fold higher affinity than that of the heterologous peptide. In addition, IGF-1 activity is also regulated by binding to specific IGF-binding proteins that do not bind insulin (
      • Clemmons D.R.
      ). IGF-1, the IGF-1 receptor, and the binding proteins are expressed in many tissues, creating an autocrine-paracrine hormonal system. One of the earliest steps in signal transduction by both insulin and IGF-1 is the extensive phosphorylation of IRS-1, a 185-kDa protein. Tyrosyl-phosphorylated IRS-1 then interacts with numerous SH2 domain-containing proteins, including PI3′-kinase and the guanine-nucleotide exchange factor Grb2/SOS. PI3′-kinase then initiates phospholipid turnover, and Grb2/SOS activation results in initiation of the MAP kinase signal transduction cascade by sequential phosphorylation and activation of proto-oncogenes Ras and Raf and the MAPK/ERK kinases (MEK1 and MEK2).
      While the effectiveness of IGF-1 on inhibition of apoptosis is well established, the signaling pathways leading to apoptosis and the mechanisms of action by which IGF-1 and other agents prevent apoptosis are largely unknown. The PI3′-kinase inhibitor wortmannin is known to block the protective action of NGF and platelet-derived growth factor in serum-deprived PC12 cells (
      • Yao R.
      • Cooper G.M.
      ); this finding suggests an important role of the PI3′-kinase pathway in apoptosis prevention by growth factors. PI3′-kinase is an important component of intracellular signal transduction processes linked directly or indirectly to diverse receptor types. PI3′-kinase is an heterodimer composed of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (
      • Carpenter C.L.
      • Duckworth B.C.
      • Auger K.R.
      • Cohen B.
      • Schaffhausen B.S.
      • Cantley L.C.
      ). The p85 subunit, through its SH2 domains, mediates the association of p110 with activated protein tyrosine kinase receptors or phosphotyrosine-containing peptides such as IRS-1 (
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ).
      In the present work, the involvement of PI3′-kinase in mediating IGF-1 prevention of apoptosis in PC12 cells was further explored using the PI3′-kinase inhibitors wortmannin and LY294002, and PC12 cells were stably transfected with either a wild-type p85 (Wp85) construct or a dominant-negative p85 construct (δp85) (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • Hawkins P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ). The δp85 protein lacks the inter-SH2 region required for binding to p110, thereby blocking activation of p110 (
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ). Rapamycin, which inhibits pp70 S6 kinase (pp70S6K) activation, was also used (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ). In addition, the involvement of the MAP kinase pathway in the prevention of apoptosis by IGF-1 was tested using PD098059, a specific inhibitor of MEK and the MAP kinase cascade (
      • Allesi D.R.
      • Cuenda A.
      • Cohen P.
      • Dudley D.T.
      • Saltiel A.R.
      ,
      • Pang L.
      • Sawada T.
      • Decker S.J.
      • Saltiel A.R.
      ).

      DISCUSSION

      During embryonic development, rapid cellular proliferation is necessary for organ growth. However, appropriate function of each organ also depends on an orderly removal of certain cells. This is achieved by the process of apoptosis, also known as programmed cell death. Apoptosis is detected initially as internucleosomal fragmentation of genomic DNA, followed by chromatin condensation, nuclear disintegration, and cellular fragmentation (
      • Kerr J.F.R.
      • Wylie A.H.
      • Currie A.R.
      ,
      • Wylie A.H.
      • Kerr J.F.R.
      • Currie A.R.
      ). In addition, apoptosis may play an important role in regulating tumorigenesis. For example, the tumor suppressor gene product, p53, may regulate tumorigenesis by inducing apoptosis in the presence of oncogenic factors (
      • Symonds H.
      • Krall L.
      • Remington L.
      • Saenz-Rubles M.
      • Lowe S.
      • Jacks T.
      • Van Dyke T.
      ). Mutations in the p53 gene are found in patients with a variety of malignancies, and a poor clinical prognosis is often correlated with the existence of these mutations. Therefore, understanding the mechanisms controlling apoptosis should enhance our ability to treat many diseases.
      Insulin and the IGFs have been shown in a number of systems to inhibit apoptosis. In cerebellar granule neurons cultured in low potassium or PC12 cells following NGF withdrawal, these growth factors inhibit apoptosis (
      • Yao R.
      • Cooper G.M.
      ,
      • D'Mello S.R.
      • Galli C.
      • Ciotti T.
      • Calissano P.
      ,
      • Galli C.
      • Meucci O.
      • Scorziello A.
      • Werge T.M.
      • Calissano P.
      • Schettini G.
      ). IGF-1 is a survival factor for neuroglial cells following ischemia (
      • Gluckman P.
      • Klempt N.
      • Guan J.
      • Mallard C.
      • Sirimanne E.
      • Dragunow M.
      • Klempt N.
      • Singh K.
      • Williams C.
      • Nikolics K.
      ). Similarly, c-myc-induced apoptosis can be prevented by IGF-1 (
      • Evan G.I.
      • Wyllie A.H.
      • Gilbert C.S.
      • Littlewood T.D.
      • Land H.
      • Brooks M.
      • Waters C.M.
      • Penn L.Z.
      • Hancock D.C.
      ,
      • Harrington E.A.
      • Bennett M.R.
      • Fanidi A.
      • Evan G.I.
      ). Induction of apoptosis in a human colorectal carcinoma cell line is inhibited by IGF-1 (
      • Wu X.
      • Fan Z.
      • Masui H.
      • Rosen N.
      • Mendelsohn J.
      ). Conversely, a reduction in the expression of the IGF-1 receptors in C6 rat glioblastoma cells using antisense strategies enhanced apoptosis and delayed tumor growth (
      • Resnicoff M.
      • Abraham D.
      • Yutanawiboonchai W.
      • Rotman H.L.
      • Kajstura J.
      • Rubin R.
      • Zoltick P.
      • Baserga R.
      ).
      While the importance of the IGF system in regulating cellular proliferation and apoptosis is well established, the cellular mechanisms involved in these processes are as yet undefined. We have begun to investigate these pathways using differentiated PC12 cells that undergo apoptosis following withdrawal of NGF from the culture medium. In these cells, we demonstrate that IGF-1 inhibits apoptosis at physiological concentrations, whereas much higher concentrations of insulin are required for comparable effects. Similar results using insulin to inhibit apoptosis have been previously described (
      • Yao R.
      • Cooper G.M.
      ,
      • Xia Z.
      • Dickens M.
      • Raingeaud J.
      • Davis R.J.
      • Greenberg M.E.
      ). This finding suggests that insulin exerts its effect via the IGF-1 receptor. However, since the number of IGF-1 receptors in PC12 cells is almost 5-fold greater than that of the insulin receptor, we cannot rule out the possibility of the insulin receptor being effective.
      Wortmannin, a fungal metabolite, demonstrates a substantial degree of specificity for PI3′-kinase compared with a number of other lipid kinases, especially when wortmannin is used at low concentrations (10−9 or 10−8M) (
      • Stephens L.
      • Eguinoa A.
      • Corey S.
      • Jackson T.
      • Hawkins P.T.
      ). PI3′-kinase comprises an 85-kDa regulatory subunit and a 110-kDa catalytic subunit that phosphorylates phosphatidylinositol at the D-3-hydroxyl of the inositol ring. Wortmannin binds irreversibly to the catalytic subunit (p110), thereby inhibiting the signaling pathway following PI3′-kinase activation. Wortmannin has previously been shown to completely inhibit PI3′-kinase activity in PC12 cells at 10−7M, with a half-maximal dose (IC50) of approximately 3 × 10−9M (
      • Yao R.
      • Cooper G.M.
      ,
      • Kimura K.
      • Hattori S.
      • Kabuyama Y.
      • Shizawa Y.
      • Takayanagi J.
      • Nakamura S.
      • Toki S.
      • Matsuda Y.
      • Onodera K.
      • Fukui Y.
      ). Due to the instability of the inhibitor in the medium, the peak inhibitory effect of wortmannin takes place after 3-4 h of incubation in the cells (
      • Kimura K.
      • Hattori S.
      • Kabuyama Y.
      • Shizawa Y.
      • Takayanagi J.
      • Nakamura S.
      • Toki S.
      • Matsuda Y.
      • Onodera K.
      • Fukui Y.
      ). In the present study, wortmannin at low concentrations enhanced apoptosis and inhibited the effect of IGF-1 when added to PC12 cells, suggesting that PI3′-kinase is involved in the regulation of apoptosis. Support for this hypothesis comes from the use of the synthetic PI3′-kinase inhibitor LY294002 (
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ), which gave essentially the same results as wortmannin. In both cases, detection of characteristic DNA laddering coincided with a 20-30% decrease in the number of cells attached to the plate and with an even more marked decrease in mitochondrial activity as measured by MTT assay. The acceleration of apoptosis by addition of wortmannin to cells in the absence of growth factor has been observed previously (
      • Scheid M.P.
      • Lauener R.W.
      • Duronio V.
      ) and can be attributed to the inhibition of the basal PI3′-kinase activity.
      Since wortmannin (and LY294002) may not be absolutely specific for PI3′-kinase, we chose to further modulate the PI3′-kinase system by stably transfecting PC12 cells with a dominant-negative p85 or a wild-type p85 construct. Overexpression of these proteins has enabled us to confirm the role of PI3′-kinase in apoptosis. Overexpression of wild-type p85 did not significantly affect IGF-1 effects on apoptosis following NGF withdrawal. The dominant-negative p85 subunit, on the other hand, can bind phosphorylated IRS-1 but, because it lacks the inter-SH2 domain, cannot bind the p110 subunit. Therefore, PI3′-kinase activation by IGF-1 is effectively inhibited by blocking access of functional p85 to phosphorylated IRS-1 (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • Hawkins P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ). Overexpression of δp85 thus resulted in a partial decrease of PI3′-kinase activity and a consequent increase in the degree of apoptosis observed in serum-free medium and an increased sensitivity to the presence of wortmannin or LY294002. Thus, our results support the findings of Yao and Cooper (
      • Yao R.
      • Cooper G.M.
      ) who similarly demonstrated the importance of PI3′-kinase in mediating the NGF modulation of apoptosis in PC12 cells. The involvement of the PI3′-kinase pathway on prevention of apoptosis by IGF-1, but not by IL-3, was also recently observed in hemopoietic progenitor cells (
      • Minshall C.
      • Arkins S.
      • Freund G.G.
      • Kelley K.W.
      ). The effect of rapamycin, an inhibitor of pp70S6K (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ), further supports the role of the PI3′-kinase pathway in mediating the IGF-1 effect on apoptosis.
      However, we have noted that overexpression of δp85 did not result in a complete inhibition of the IGF-1 action, suggesting the possibility that IGF-1 may inhibit apoptosis via other signal transduction pathways. We chose, therefore, to study the role of the MAP kinase pathway using a synthetic inhibitor of MEK. The MAP kinase pathway is responsible for mediating numerous effects of both insulin and IGF-1. Both insulin and IGF-1 receptors activate the MAP kinase cascade through a p21ras-dependent signal transduction pathway. Activated Ras interacts with the serine-threonine kinase Raf and localizes it to the membrane, thereby initiating Raf activation. Activated Raf then initiates the kinase cascade by phosphorylating and activating MEK, which in turn phosphorylates and activates the MAPK/ERK.
      PD098059 is a small molecular weight inhibitor of MEK activity, as measured by MAP kinase activity (
      • Allesi D.R.
      • Cuenda A.
      • Cohen P.
      • Dudley D.T.
      • Saltiel A.R.
      ,
      • Pang L.
      • Sawada T.
      • Decker S.J.
      • Saltiel A.R.
      ). PD098059 is a specific noncompetitive inhibitor of MEK, with respect to ATP binding, and does not inhibit several other kinases tested (
      • Allesi D.R.
      • Cuenda A.
      • Cohen P.
      • Dudley D.T.
      • Saltiel A.R.
      ). PD098059 inhibits MAP kinase activity in PC12 cells with an IC50 of approximately 10−6M (
      • Allesi D.R.
      • Cuenda A.
      • Cohen P.
      • Dudley D.T.
      • Saltiel A.R.
      ,
      • Pang L.
      • Sawada T.
      • Decker S.J.
      • Saltiel A.R.
      ). In our study, incubation of PC12 cells with concentrations of PD098059 capable of significantly inhibiting MAP kinase activity resulted in increased apoptosis when given alone. PD098059 could also block the protective effect of IGF-1. After 6 h of incubation with PD098059, neither a decrease in cell number nor DNA laddering could be found although a decrease in the metabolic activity in the cells treated with the inhibitor could be already detected by this time. This decrease in mitochondrial activity precedes by some hours the initiation of apoptosis as detected by loss of cell number and the appearance of DNA laddering. This loss of metabolic activity preceding apoptosis triggering has been previously found in sympathetic neurons deprived of growth factor (
      • Deckwerth T.L.
      • Johnson Jr., E.M.
      ). Thus, our results demonstrate that the MAP kinase pathway is also involved in IGF-1 prevention of apoptosis. A different conclusion was reached by Yao and Cooper (
      • Yao R.
      • Cooper G.M.
      ) using PC12 cells. However, their studies involved the use of the expression of the dominant inhibitory mutant Ras N17, which interferes with normal Ras function and Raf activation. Since Raf and MEK activation may be achieved by pathways other than Ras (
      • Cross D.A.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Karnitz L.M.
      • Burns L.A.
      • Sutor S.L.
      • Blenis J.
      • Abraham R.T.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ), we suggest that inhibition of the pathway downstream of Raf, as in the present study, is important to establish the role of the MAP kinase pathway. Moreover, in PC12 cells, it has been described that the expression of an activated Raf mutant did not activate the MAP kinases although it resulted in gene expression similar to that induced by NGF (
      • Wood K.
      • Qi H.
      • D'Arcangelo G.
      • Armstrong R.
      • Roberts T.
      • Halegoua S.
      ). This result suggests that the MAP kinases may be activated by other pathways in addition to the Ras/Raf pathway in this cell type. Further confirmation for our conclusion that the MAP kinase pathway is involved in IGF-1 inhibition of apoptosis comes from a recent study (
      • Xia Z.
      • Dickens M.
      • Raingeaud J.
      • Davis R.J.
      • Greenberg M.E.
      ) that demonstrates a role for the MAP kinases in apoptosis prevention by NGF in PC12 cells.
      Since the PI3′-kinase and MAP kinase pathways converge at some point before MEK activation (
      • Cross D.A.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Karnitz L.M.
      • Burns L.A.
      • Sutor S.L.
      • Blenis J.
      • Abraham R.T.
      ), it is possible that the wortmannin inhibition of PI3′-kinase in our cells affects MAP kinase activation. Furthermore, recent studies have also shown that following certain stimuli, MEK may be activated by a PI3′-kinase-dependent pathway (
      • Karnitz L.M.
      • Burns L.A.
      • Sutor S.L.
      • Blenis J.
      • Abraham R.T.
      ). Consistent with this are our findings that wortmannin is also capable of inhibiting IGF-1-stimulated MAP kinase activity to a certain extent. However, while the possibility that the wortmannin effect is entirely due to the inhibition of the MAP kinase pathway is consistent with our data, it is less likely because the wortmannin effect on IGF-1 inhibition of apoptosis occurs much earlier than the effect of PD098059 on IGF-1 action. Furthermore, rapamycin alone was also capable of inhibiting the effect of IGF-1 in preventing apoptosis. We therefore postulate that both pathways are involved separately in this process and that the synergistic effect we obtained by using the MEK inhibitor together with wortmannin suggests some convergence of the two pathways either at the level of MEK or at a more distal point. The convergence of the two pathways is supported by our findings that the effectiveness of wortmannin was greater than the effect of rapamycin.
      While we have described in this study two separate IGF-1-stimulated pathways that inhibit apoptosis, it is also likely that IGF-1 inhibition of apoptosis involves other as yet unidentified pathways. The elucidation of these pathways should ultimately lead to a better understanding of cellular growth and apoptosis and enable investigators to design new and effective therapeutic agents.

      Acknowledgments

      We thank Professor Masato Kasuga (Kobe University, Japan) for the gifts of the wild-type and dominant-negative p85 constructs and Drs. V. Blakesley and E. Wertheimer (National Institutes of Health, Bethesda, MD) for critical reading of the manuscript.

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