Showing papers on "PI3K/AKT/mTOR pathway published in 2000"

Modulation of Hypoxia-inducible Factor 1α Expression by the Epidermal Growth Factor/Phosphatidylinositol 3-Kinase/PTEN/AKT/FRAP Pathway in Human Prostate Cancer Cells: Implications for Tumor Angiogenesis and Therapeutics

Abstract: Dysregulated signal transduction from receptor tyrosine kinases to phosphatidylinositol 3-kinase (PI3K), AKT (protein kinase B), and its effector FKBP-rapamycin-associated protein (FRAP) occurs via autocrine stimulation or inactivation of the tumor suppressor PTEN in many cancers. Here we demonstrate that in human prostate cancer cells, basal-, growth factor-, and mitogen-induced expression of hypoxia-inducible factor 1 (HIF-1) alpha, the regulated subunit of the transcription factor HIF-1, is blocked by LY294002 and rapamycin, inhibitors of PI3K and FRAP, respectively. HIF-1-dependent gene transcription is blocked by dominant-negative AKT or PI3K and by wild-type PTEN, whereas transcription is stimulated by constitutively active AKT or dominant-negative PTEN. LY294002 and rapamycin also inhibit growth factor- and mitogen-induced secretion of vascular endothelial growth factor, the product of a known HIF-1 target gene, thus linking the PI3K/PTEN/AKT/FRAP pathway, HIF-1, and tumor angiogenesis. These data indicate that pharmacological agents that target PI3K, AKT, or FRAP in tumor cells inhibit HIF-1alpha expression and that such inhibition may contribute to therapeutic efficacy.

A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells

Abstract: The microbially derived antiproliferative agent rapamycin inhibits cell growth by interfering with the signaling functions of the mammalian target of rapamycin (mTOR). In this study, we demonstrate that interleukin-3 stimulation induces a wortmannin-sensitive increase in mTOR kinase activity in a myeloid progenitor cell line. The involvement of phosphoinositide 3'-kinase (PI3K) in the regulation of mTOR activity was further suggested by findings that mTOR was phosphorylated in vitro and in vivo by the PI3K-regulated protein kinase, AKT/PKB. Although AKT phosphorylated mTOR at two COOH-terminal sites (Thr2446 and Ser2448) in vitro, Ser2448 was the major phosphorylation site in insulin-stimulated or -activated AKT-expressing human embryonic kidney cells. Transient transfection assays with mTOR mutants bearing Ala substitutions at Ser2448 and/or Thr2446 indicated that AKT-dependent mTOR phosphorylation was not essential for either PHAS-I phosphorylation or p70S6K activation in HEK cells. However, a deletion of amino acids 2430-2450 in mTOR, which includes the potential AKT phosphorylation sites, significantly increased both the basal protein kinase activity and in vivo signaling functions of mTOR. These results demonstrate that mTOR is a direct target of the PI3K-AKT signaling pathway in mitogen-stimulated cells, and that the identified AKT phosphorylation sites are nested within a "repressor domain" that negatively regulates the catalytic activity of mTOR. Furthermore, the activation status of the PI3K-AKT pathway in cancer cells may be an important determinant of cellular sensitivity to the cytostatic effect of rapamycin.

Roles for Insulin Receptor, PI3-Kinase, and Akt in Insulin-Signaling Pathways Related to Production of Nitric Oxide in Human Vascular Endothelial Cells

Abstract: Background—Previously, we demonstrated that insulin stimulates production of nitric oxide (NO) in endothelial cells. However, specific insulin-signaling pathways mediating production of NO have not been elucidated. Methods and Results—We developed methods for transfection of human umbilical vein endothelial cells (HUVECs) and direct measurement of NO to begin defining insulin-signaling pathways related to NO production. HUVECs were cotransfected with enhanced Green Fluorescent Protein (eGFP) and another gene of interest. Transfection efficiencies >95% were obtained by selecting cells expressing eGFP. Overexpression of insulin receptors in HUVECs resulted in an ≈3-fold increase in production of NO in response to insulin. In contrast, HUVECs overexpressing a tyrosine kinase–deficient mutant insulin receptor had a dose-response curve similar to that of control cells. Overexpression of inhibitory mutants of either phosphatidylinositol 3-kinase (PI3K) or Akt resulted in nearly complete inhibition of insulin-st...

Akt suppresses apoptosis by stimulating the transactivation potential of the RelA/p65 subunit of NF-κB

Abstract: It is well established that cell survival signals stimulated by growth factors, cytokines, and oncoproteins are initiated by phosphoinositide 3-kinase (PI3K)- and Akt-dependent signal transduction pathways. Oncogenic Ras, an upstream activator of Akt, requires NF-kappaB to initiate transformation, at least partially through the ability of NF-kappaB to suppress transformation-associated apoptosis. In this study, we show that oncogenic H-Ras requires PI3K and Akt to stimulate the transcriptional activity of NF-kappaB. Activated forms of H-Ras and MEKK stimulate signals that result in nuclear translocation and DNA binding of NF-kappaB as well as stimulation of the NF-kappaB transactivation potential. In contrast, activated PI3K or Akt stimulates NF-kappaB-dependent transcription by stimulating transactivation domain 1 of the p65 subunit rather than inducing NF-kappaB nuclear translocation via IkappaB degradation. Inhibition of IkappaB kinase (IKK), using an IKKbeta dominant negative protein, demonstrated that activated Akt requires IKK to efficiently stimulate the transactivation domain of the p65 subunit of NF-kappaB. Inhibition of endogenous Akt activity sensitized cells to H-Ras(V12)-induced apoptosis, which was associated with a loss of NF-kappaB transcriptional activity. Finally, Akt-transformed cells were shown to require NF-kappaB to suppress the ability of etoposide to induce apoptosis. Our work demonstrates that, unlike activated Ras, which can stimulate parallel pathways to activate both DNA binding and the transcriptional activity of NF-kappaB, Akt stimulates NF-kappaB predominantly by upregulating of the transactivation potential of p65.

Toll-like receptor 2-mediated NF-kappa B activation requires a Rac1-dependent pathway.

Abstract: Mammalian Toll-like receptors (TLRs) are expressed on innate immune cells and respond to the membrane components of Gram-positive or Gram-negative bacteria. When activated, they convey signals to transcription factors that orchestrate the inflammatory response. However, the intracellular signaling events following TLR activation are largely unknown. Here we show that TLR2 stimulation by Staphylococcus aureus induces a fast and transient activation of the Rho GTPases Rac1 and Cdc42 in the human monocytic cell line THP-1 and in 293 cells expressing TLR2. Dominant-negative Rac1N17, but not dominant-negative Cdc42N17, block nuclear factor-κB (NF-κB) transactivation. S. aureus stimulation causes the recruitment of active Rac1 and phosphatidylinositol-3 kinase (PI3K) to the TLR2 cytosolic domain. Tyrosine phosphorylation of TLR2 is required for assembly of a multiprotein complex that is necessary for subsequent NF-κB transcriptional activity. A signaling cascade composed of Rac1, PI3K and Akt targets nuclear p65 transactivation independently of IκBα degradation. Thus Rac1 controls a second, IκB–independent, pathway to NF-κB activation and is essential in innate immune cell signaling via TLR2.

Regulation of cellular growth by the Drosophila target of rapamycin dTOR

Abstract: The TOR protein kinases (TOR1 and TOR2 in yeast; mTOR/FRAP/RAFT1 in mammals) promote cellular proliferation in response to nutrients and growth factors, but their role in development is poorly understood. Here, we show that the Drosophila TOR homolog dTOR is required cell autonomously for normal growth and proliferation during larval development, and for increases in cellular growth caused by activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. As in mammalian cells, the kinase activity of dTOR is required for growth factor-dependent phosphorylation of p70 S6 kinase (p70S6K) in vitro, and we demonstrate that overexpression of p70S6K in vivo can rescue dTOR mutant animals to viability. Loss of dTOR also results in cellular phenotypes characteristic of amino acid deprivation, including reduced nucleolar size, lipid vesicle aggregation in the larval fat body, and a cell type-specific pattern of cell cycle arrest that can be bypassed by overexpression of the S-phase regulator cyclin E. Our results suggest that dTOR regulates growth during animal development by coupling growth factor signaling to nutrient availability.

Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1).

Abstract: Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27KIP1 through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27KIP1 promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27KIP1 appears to be critical in the regulation of cell survival since mere ectopic expression of p27KIP1 was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27KIP1 null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27KIP1 transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

The rapamycin-sensitive signal transduction pathway as a target for cancer therapy

Abstract: The high frequency of mutations in cancer cells which result in altered cell cycle regulation and growth signal transduction, conferring a proliferative advantage, indicates that many of these aberrant mechanisms may be strategic targets for cancer therapy. The macrolide fungicide rapamycin, a natural product with potent antimicrobial, immunosuppressant, and anti-tumor properties, inhibits the translation of key mRNAs of proteins required for cell cycle progression from G1 to S phase. Rapamycin binds intracellularly to the immunophilin FK506 binding protein 12 (FKBP12), and the resultant complex inhibits the protein kinase activity of a protein kinase termed mammalian target of rapamycin (mTOR). The inhibition of mTOR, in turn, blocks signals to two separate downstream pathways which control the translation of specific mRNAs required for cell cycle traverse from G1 to S phase. Blocking mTOR affects the activity of the 40S ribosomal protein S6 kinase (p70s6k) and the function of the eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), leading to growth arrest in the the G1 phase of the cell cycle. In addition to its actions on p70s6k and 4E-BP1, rapamycin prevents cyclin-dependent kinase activation, inhibits retinoblastoma protein (pRb) phosphorylation, and accelerates the turnover of cyclin D1 that leads to a deficiency of active cdk4/cyclin D1 complexes, all of which can inhibit cell cycle traverse at the G1/S phase transition. Both rapamycin and CCI-779, an ester analog of rapamycin with improved pharmaceutical properties and aqueous solubility, have demonstrated impressive activity against a broad range of human cancers growing in tissue culture and in human tumor xenograft models, which has supported the development of compounds targeting rapamycin-sensitive signal-transduction pathways. CCI-779 has completed several phase I clinical evaluations and is currently undergoing broad disease-directed efficacy studies. The agent appears to be well tolerated at doses that have resulted in impressive anti-tumor activity in several types of refractory neoplasms. Important challenges during clinical development include the definition of a recommended dose range associated with optimal biological activity and maximal therapeutic indices, as well as the ability to predict which tumors will be sensitive or resistant to CCI-779.

The conserved phosphoinositide 3-kinase pathway determines heart size in mice

Abstract: Phosphoinositide 3-kinase (PI3K) has been shown to regulate cell and organ size in Drosophila, but the role of PI3K in vertebrates in vivo is not well understood. To examine the role of PI3K in intact mammalian tissue, we have created and characterized transgenic mice expressing constitutively active or dominant-negative mutants of PI3K in the heart. Cardiac- specific expression of constitutively active PI3K resulted in mice with larger hearts, while dominant-negative PI3K resulted in mice with smaller hearts. The increase or decrease in heart size was associated with comparable increase or decrease in myocyte size. Cardiomyopathic changes, such as myocyte necrosis, apoptosis, interstitial fibrosis or contractile dysfunction, were not observed in either of the transgenic mice. Thus, the PI3K pathway is necessary and sufficient to promote organ growth in mammals.

Multiple pathways control protein kinase C phosphorylation.

Abstract: The protein kinase C (PKC) family of signal transducers are characterized by a dependence upon lipids for activity. Specifically, the classical (cPKCα, β and γ) and novel (nPKCδ, ϵ, η and θ) PKC isotypes display a physiological requirement for diacylglycerol for activity. This property of PKC has defined a now well established signalling pathway operating through receptors to phosphatidylinositol‐specific phospholipase C and hence via diacylglycerol (DAG) [and inositol (1,4,5) trisphosphate Ins(1,4,5)P3/Ca2+] to PKC (Figure 1). The operation of this pathway has been described in many cell types, and numerous reviews have covered this signalling paradigm (see Nishizuka, 1986; Hug et al ., 1993; Dekker and Parker, 1994; Jaken, 1996). Figure 1. The classical pathway of PKC activation. The scheme illustrates the production of the immediate precursor lipid PtdIns(4,5)P2 from its parent lipid PtdIns. Various agonists are linked to the phospholipases (PtdIns‐PLC) that can cleave PtdIns(4,5)P2 to diacylglycerol (DAG) and the calcium mobilizer Ins(1,4,5)P3. Calcium can affect the cPKC class by promoting membrane recruitment, but the key allosteric activator at the membrane for both cPKC and nPKC isotypes is DAG. More recently, attention has been drawn to the phosphorylation of PKC itself. Intriguingly, what was once considered a purely effector‐driven transducer turns out to possess a complex amplitude control. The elucidation of this phosphorylation control in the cPKC isotypes has formed the basis for understanding the behaviour of the immediate family, with implications for other related AGC kinases (see Hanks and Hunter, 1995; further information is available at the Protein Kinase Resource: http://www.sdsc.edu/Kinases). The further characterization of the kinases that act upon the nPKCs provides evidence of three distinct input pathways converging upon PKC. Thus, PKC serves as an elegant example of the manner in which multiple signals are integrated …

Membrane Estrogen Receptor Engagement Activates Endothelial Nitric Oxide Synthase via the PI3-Kinase–Akt Pathway in Human Endothelial Cells

Abstract: —17β-Estradiol (E2) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E2-stimulated endothelial NO release can occur without an increase in cytosolic Ca2+. Here we demonstrate for the first time, to our knowledge, that E2 rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase–Akt pathway. E2 treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182,780. E2 stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E2-activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca2+ levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E2-stimulated NO release. The membrane-impermeant E2BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase–Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca2+ fluxes and describes an important pathway relevant to cardiovascular pathophysiology.

Forkhead Transcription Factors Are Critical Effectors of Cell Death and Cell Cycle Arrest Downstream of PTEN

Abstract: PTEN acts as a tumor suppressor, at least in part, by antagonizing phosphoinositide 3-kinase (PI3K)/Akt signaling. Here we show that Forkhead transcription factors FKHRL1 and FKHR, substrates of the Akt kinase, are aberrantly localized to the cytoplasm and cannot activate transcription in PTEN-deficient cells. Restoration of PTEN function restores FKHR to the nucleus and restores transcriptional activation. Expression of a constitutively active form of FKHR that cannot be phosphorylated by Akt produces the same effect as reconstitution of PTEN on PTEN-deficient tumor cells. Specifically, activated FKHR induces apoptosis in cells that undergo PTEN-mediated cell death and induces G(1) arrest in cells that undergo PTEN-mediated cell cycle arrest. Furthermore, both PTEN and constitutively active FKHR induce p27(KIP1) protein but not p21. These data suggest that Forkhead transcription factors are critical effectors of PTEN-mediated tumor suppression.

HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the Akt pathway.

Abstract: HER-2/neu has been implicated in the activation of androgen receptor (AR) and in inducing hormone-independent prostate cancer growth. Here we report that HER-2/neu activates Akt (protein kinase B) to promote prostate cancer cell survival and growth in the absence of androgen. Blocking of the Akt pathway by a dominant-negative Akt or an inhibitor LY294002 abrogates the HER-2/neu-induced AR signaling and cell survival/growth effects in the absence or presence of androgen. Akt specifically binds to AR and phosphorylates serines 213 and 791 of AR. Thus, Akt is a novel activator of AR required for HER-2/neu signaling to androgen-independent survival and growth of prostate cancer cells.

Akt regulates cell survival and apoptosis at a postmitochondrial level.

Abstract: Phosphoinositide 3 kinase/Akt pathway plays an essential role in neuronal survival. However, the cellular mechanisms by which Akt suppresses cell death and protects neurons from apoptosis remain unclear. We previously showed that transient expression of constitutively active Akt inhibits ceramide-induced death of hybrid motor neuron 1 cells. Here we show that stable expression of either constitutively active Akt or Bcl-2 inhibits apoptosis, but only Bcl-2 prevents the release of cytochrome c from mitochondria, suggesting that Akt regulates apoptosis at a postmitochondrial level. Consistent with this, overexpressing active Akt rescues cells from apoptosis without altering expression levels of endogenous Bcl-2, Bcl-x, or Bax. Akt inhibits apoptosis induced by microinjection of cytochrome c and lysates from cells expressing active Akt inhibit cytochrome c induced caspase activation in a cell-free assay while lysates from Bcl-2–expressing cells have no effect. Addition of cytochrome c and dATP to lysates from cells expressing active Akt do not activate caspase-9 or -3 and immunoprecipitated Akt added to control lysates blocks cytochrome c–induced activation of the caspase cascade. Taken together, these data suggest that Akt inhibits activation of caspase-9 and -3 by posttranslational modification of a cytosolic factor downstream of cytochrome c and before activation of caspase-9.

Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer

Abstract: We previously demonstrated that AKT2, a member of protein kinase B family, is activated by a number of growth factors via Ras and PI 3-kinase signaling pathways. Here, we report the frequent activation of AKT2 in human primary ovarian cancer and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase (PI 3-kinase)/Akt pathway. In vitro AKT2 kinase assay analyses in 91 ovarian cancer specimens revealed elevated levels of AKT2 activity (>3-fold) in 33 cases (36.3%). The majority of tumors displaying activated AKT2 were high grade and stages III and IV. Immunostaining and Western blot analyses using a phospho-ser-473 Akt antibody that detects the activated form of AKT2 (AKT2 phosphorylated at serine-474) confirmed the frequent activation of AKT2 in ovarian cancer specimens. Phosphorylated AKT2 in tumor specimens localized to the cell membrane and cytoplasm but not the nucleus. To address the mechanism of AKT2 activation, we measured in vitro PI 3-kinase activity in 43 ovarian cancer specimens, including the 33 cases displaying elevated AKT2 activation. High levels of PI 3-kinase activity were observed in 20 cases, 15 of which also exhibited AKT2 activation. The remaining five cases displayed elevated AKT1 activation. Among the cases with elevated AKT2, but not PI 3-kinase activity (18 cases), three showed down-regulation of PTEN protein expression. Inhibition of PI 3-kinase/AKT2 by wortmannin or LY294002 induces apoptosis in ovarian cancer cells exhibiting activation of the PI 3-kinase/AKT2 pathway. These findings demonstrate for the first time that activation of AKT2 is a common occurrence in human ovarian cancer and that PI 3-kinase/Akt pathway may be an important target for ovarian cancer intervention.

Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation.

Abstract: Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P2 and PI-3,4,5-P3 in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-γ1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.

Protein kinases as mediators of phosphoinositide 3-kinase signaling.

Abstract: Phosphoinositide 3-kinase (PI3K) is an enzyme that participates in a myriad of cellular processes and whose activity has been linked to cell growth and transformation, differentiation, motility, insulin action, and cell survival to name a few. Direct links between PI3K action and human diseases have

Inhibition of phosphatidylinositide 3-kinase enhances gemcitabine-induced apoptosis in human pancreatic cancer cells.

Abstract: Human pancreatic adenocarcinoma cell lines PK1 and PK8 are resistant to the clinically relevant chemotherapy agent gemcitabine. Cell cycle analysis demonstrated an accumulation of cells in the early S phase during treatment with 20 microM gemcitabine, consistent with its mode of action as a DNA chain terminator. However, apoptosis was evident in only a small percentage of cells. Similar to pancreatic cancers in the clinic, PK1 and PK8 cells carry constitutively active Ki-Ras and overexpress multiple receptor tyrosine kinases. Both genetic abnormalities may potentially up-regulate the activity of the phosphatidylinositide 3-kinase (P13K)-protein kinase B (PKB)/Akt cell survival pathway. The current study examined the relevance of this pathway in the modulation of drug resistance in PK1 and PK8 cells. After exposure to 20 microM gemcitabine for 48 h and in the continuous presence of the drug, treatment with the P13K inhibitors wortmannin (50-200 nM) and LY294002 (15-120 microM) for 4 h substantially enhanced apoptosis in a concentration-dependent manner as compared with treatment with gemcitabine alone, as determined by the loss of mitochondrial membrane potential and the increase in propidium iodide uptake using flow cytometry. Furthermore, Western blotting showed that the reduction of phosphorylated PKB/Akt levels correlated with the enhancement of gemcitabine-induced apoptosis, suggesting that the PI3K-PKB/Akt pathway plays a significant role in mediating drug resistance in human pancreatic cancer cells. PI3K inhibitors may have therapeutic potential when combined with gemcitabine in the treatment of pancreatic cancers.

Granulocyte-Macrophage Colony-Stimulating Factor Delays Neutrophil Constitutive Apoptosis Through Phosphoinositide 3-Kinase and Extracellular Signal-Regulated Kinase Pathways

Abstract: Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0. 0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways.

Akt/Protein Kinase B Prevents Injury-Induced Motoneuron Death and Accelerates Axonal Regeneration

Abstract: Motoneurons require neurotrophic factors for their survival and axonal projection during development, as well as nerve regeneration. By using the axotomy-induced neuronal death paradigm and adenovirus-mediated gene transfer, we attempted to gain insight into the functional significances of major growth factor receptor downstream cascades, Ras–extracellular signal-regulated kinase (Ras-ERK) pathway and phosphatidylinositol-3 kinase–Akt (PI3K-Akt) pathway. After neonatal hypoglossal nerve transection, the constitutively active Akt-overexpressing neurons could survive as well as those overexpressing Bcl-2, whereas the constitutively active ERK kinase (MEK)-overexpressing ones failed to survive. A dominant negative Akt experiment demonstrated that inhibition of Akt pathway hastened axotomy-induced neuronal death in the neonate. In addition, the dominant active Akt-overexpressing adult hypoglossal neurons showed accelerated axonal regeneration after axotomy. These results suggest that Akt plays dual roles in motoneuronal survival and nerve regeneration in vivo and that PI3K-Akt pathway is probably more vital in neuronal survival after injury than Ras-ERK pathway.

New Role for Shc in Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

Abstract: Most, if not all, cytokines activate phosphatidylinositol 3-kinase (PI-3K) Although many cytokine receptors have direct binding sites for the p85 subunit of PI-3K, others, such as the interleukin-3 (IL-3) receptor beta common chain (βc) and the IL-2 receptor beta chain (IL-2Rβ), lack such sites, leaving the mechanism by which they activate PI-3K unclear Here, we show that the protooncoprotein Shc, which promotes Ras activation by recruiting the Grb2-Sos complex in response to stimulation of cytokine stimulation, also signals to the PI-3K/Akt pathway Analysis of Y→F and “add-back” mutants of βc shows that Y577, the Shc binding site, is the major site required for Gab2 phosphorylation in response to cytokine stimulation When fused directly to a mutant form of IL-2Rβ that lacks other cytoplasmic tyrosines, Shc can promote Gab2 tyrosyl phosphorylation Mutation of the three tyrosyl phosphorylation sites of Shc, which bind Grb2, blocks the ability of the Shc chimera to evoke Gab2 tyrosyl phosphorylation Overexpression of mutants of Grb2 with inactive SH2 or SH3 domains also blocks cytokine-stimulated Gab2 phosphorylation The majority of cytokine-stimulated PI-3K activity associates with Gab2, and inducible expression of a Gab2 mutant unable to bind PI-3K markedly impairs IL-3-induced Akt activation and cell growth Experiments with the chimeric receptors indicate that Shc also signals to the PI-3K/Akt pathway in response to IL-2 Our results suggest that cytokine receptors lacking direct PI-3K binding sites activate Akt via a Shc/Grb2/Gab2/PI-3K pathway, thereby regulating cell survival and/or proliferation