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• W2166608001 abstract "Cancer cells in which the PTEN lipid phosphatase gene is deleted have constitutively activated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and require activation of this pathway for survival. In non-small cell lung cancer (NSCLC) cells, PI3K-dependent signaling is typically activated through mechanisms other than PTEN gene loss. The role of PI3K in the survival of cancer cells that express wild-type PTEN has not been defined. Here we provide evidence that H1299 NSCLC cells, which express wild-type PTEN, underwent proliferative arrest following treatment with an inhibitor of all isoforms of class I PI3K catalytic activity (LY294002) or overexpression of the PTEN lipid phosphatase. In contrast, overexpression of a dominant-negative mutant of the p85α regulatory subunit of PI3K (Δp85) induced apoptosis. Whereas PTEN and Δ85 both inhibited activation of AKT/protein kinase B, only Δp85 inhibited c-Jun NH2-terminal kinase (JNK) activity. Cotransfection of the constitutively active mutant Rac-1 (Val12), an upstream activator of JNK, abrogated Δp85-induced lung cancer cell death, whereas constitutively active mutant mitogen-activated protein kinase kinase (MKK)-1 (R4F) did not. Furthermore, LY294002 induced apoptosis of MKK4-null but not wild-type mouse embryo fibroblasts. Therefore, we propose that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival. Cancer cells in which the PTEN lipid phosphatase gene is deleted have constitutively activated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and require activation of this pathway for survival. In non-small cell lung cancer (NSCLC) cells, PI3K-dependent signaling is typically activated through mechanisms other than PTEN gene loss. The role of PI3K in the survival of cancer cells that express wild-type PTEN has not been defined. Here we provide evidence that H1299 NSCLC cells, which express wild-type PTEN, underwent proliferative arrest following treatment with an inhibitor of all isoforms of class I PI3K catalytic activity (LY294002) or overexpression of the PTEN lipid phosphatase. In contrast, overexpression of a dominant-negative mutant of the p85α regulatory subunit of PI3K (Δp85) induced apoptosis. Whereas PTEN and Δ85 both inhibited activation of AKT/protein kinase B, only Δp85 inhibited c-Jun NH2-terminal kinase (JNK) activity. Cotransfection of the constitutively active mutant Rac-1 (Val12), an upstream activator of JNK, abrogated Δp85-induced lung cancer cell death, whereas constitutively active mutant mitogen-activated protein kinase kinase (MKK)-1 (R4F) did not. Furthermore, LY294002 induced apoptosis of MKK4-null but not wild-type mouse embryo fibroblasts. Therefore, we propose that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival. Class I phosphatidylinositol 3-kinase (PI3K) 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-bisphosphate; PI(4,5)P, phosphatidylinositol 4,5-phosphate; PI(4)P, phosphatidylinositol 4-phosphate; MKK, mitogenactivated protein kinase kinase; JNK, c-Jun NH2-terminal kinase; NSCLC, non-small cell lung cancer; MEF, mouse embryo fibroblast; EGF, epidermal growth factor; IGF-1, insulin-like growth factor-1; MAP, mitogen-activated protein kinase; SH2, Src homology domain 2; HA, hemagglutinin; PBD, p21 binding domain; CMV, cytomegalovirus; GST, glutathione S-transferase; MBP, myelin basic protein; GSK3, glycogen synthase kinase 3; CDK2, cyclin-dependent kinase 2; ERK1/2, extracellular signal-regulated kinase 1/2; BrdUrd, bromodeoxyuridine; PTEN, phosphatase and tensin homolog deleted from chromosome 10. 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-bisphosphate; PI(4,5)P, phosphatidylinositol 4,5-phosphate; PI(4)P, phosphatidylinositol 4-phosphate; MKK, mitogenactivated protein kinase kinase; JNK, c-Jun NH2-terminal kinase; NSCLC, non-small cell lung cancer; MEF, mouse embryo fibroblast; EGF, epidermal growth factor; IGF-1, insulin-like growth factor-1; MAP, mitogen-activated protein kinase; SH2, Src homology domain 2; HA, hemagglutinin; PBD, p21 binding domain; CMV, cytomegalovirus; GST, glutathione S-transferase; MBP, myelin basic protein; GSK3, glycogen synthase kinase 3; CDK2, cyclin-dependent kinase 2; ERK1/2, extracellular signal-regulated kinase 1/2; BrdUrd, bromodeoxyuridine; PTEN, phosphatase and tensin homolog deleted from chromosome 10. consists of a family of heterodimeric complexes composed of a p110 catalytic subunit and a regulatory subunit that exists predominantly in a p85 form (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Google Scholar, 2Vanhaesbrock B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Google Scholar, 3Wymann M.P. Pirola L. Biochim. Biophys. Acta. 1998; 1436: 127-150Google Scholar). The known gene family members for p85 (α, β, and γ) and p110 (α, β, δ, and γ) are expressed in a tissue-specific fashion. p85α and -β can also exist in smaller forms (p50 and p55). PI3K phosphorylates the D3 position of PI on PI(4)P and PI(4,5)P to produce PI(3,4)P2 and PI(3,4,5)P3. The 3′ sites of PI(3,4)P2 and PI(3,4,5)P3 are dephosphorylated by the PTEN tumor suppressor, whereas the 5′ site of PI(3,4,5)P3 is dephosphorylated by SHIP to produce PI(3,4)P2 (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Google Scholar). These mechanisms tightly regulate the levels of 3-phosphorylated PI in the cell. PI(3,4,5)P3 and PI(3,4)P2 recruit the pleckstrin homology domains of specific intracellular proteins to the plasma membrane, an essential event in the activation of PI3K-dependent kinases such as phosphoinositide-dependent kinase-1 and AKT, also known as protein kinase B. In addition, AKT phosphorylation at Thr308 by phosphoinositide-dependent kinase-1 and Ser473 by integrin-linked kinase (and possibly other kinases) constitutes an essential event in AKT activation (4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R.J. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Google Scholar, 5Persad S. Attwell S. Gray V. Delcommenne M. Troussard A. Sanghera J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3207-3212Google Scholar). The PI3K pathway clearly has a key role in cellular survival and transformation. AKT phosphorylates several pro- and anti-apoptotic proteins, including the Bcl-2 family member BAD, caspase-9, cyclic AMP response element-binding protein, the inhibitor of NF-κB kinase IKKα, and forkhead transcription factor-1 (6Di Cristofano A. Pandolfi P.P. Cell. 2000; 100: 387-390Google Scholar). Tumor cells feature genetic and epigenetic alterations of p85α, p110α/β, AKT2, AKT3, and PTEN that activate PI3K-dependent signaling (7Bellacosa A. de Feo D. Godwin A.K. Bell D.W. Cheng J.Q. Altomare D.A. Wan M. Dubeau L. Scambia G. Godwin A.K. Int. J. Cancer. 1995; 64: 280-285Google Scholar, 8Cheng J.Q. Ruggeri B. Klein W.M. Sonoda G. Altomore D.A. Watson D.K. Testa J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3636-3641Google Scholar, 9Janssen J.W.G. Schleithoff L. Bartram C.R. Schultz A.S. Oncogene. 1998; 16: 1767-1772Google Scholar, 10Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Google Scholar, 11Shayesteh L.M. Lu Y. Kuo W.L. Baldocchi R. Godfrey T. Collins C. Pinkel D. Powell B. Mills G.B. Gray J.W. Nat. Genet. 1999; 21: 99-102Google Scholar, 12Steck P.A. Pershouse M.A. Jasser S.A. Yung W.K.A. Lin H. Ligon A.H. Langford L.A. Baumgard M.L. Hattier T. Davis T. Frye C. Hu R. Swedlund B. David H.F. Tavtigian T. Tavtigian S.V. Nat. Genet. 1997; 15: 356-362Google Scholar, 13Teng D.H. Hu R. Lin H. Davis T. Iliev D. Frye C. Swedlund B. Hansen K.L. Vinson V.L. Gumpper K.L. Ellis L. El-Naggar A. Frazier M. Jasser S. Langford L.A. Lee J. Mills G.B. Pershouse M.A. Pollack R.E. Tornos C. Troncoso P. Yung W.K. Fujii G. Berson A. Steck P.A. Cancer Res. 1997; 57: 5221-5225Google Scholar). In vitro studies have confirmed the oncogenic effects of PI3K and its downstream mediators as well as the tumor-suppressive properties of PTEN (14Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Google Scholar, 15Chang H.W. Aoki M. Fruman D. Auger K.R. Bellacosa A. Tsichlis P.N. Cantley L.C. Roberts T.M. Vogt P.K. Science. 1997; 276: 1848-1850Google Scholar, 16Furnari F.B. Lin H. Huang H.-J. Cavenee W.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12479-12484Google Scholar, 17Klippel A. Escobedo M.A. Wachowicz M.S. Apell G. Brown T.W. Giedlin M.A. Kavanaugh W.M. Williams L.T. Mol. Cell. Biol. 1998; 98: 5699-5711Google Scholar, 18Li D.M. Sun H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15406Google Scholar, 19Lu Y. Lin Y.Z. LaPushin R. Cuevas B. Fang X. Yu S.X. Davies M.A. Khan H. Furui T. Mao M. Zinner R. Hung M.C. Steck P. Siminovitch K. Mills G.B. Oncogene. 1999; 18: 7034-7045Google Scholar). PI3K mediates its oncogenic effects, in part, through the GTP-binding protein Rac-1, which plays a key role in the reorganization of the actin cytoskeleton induced by growth factors or oncogenic Ras (20Qiu R.G. Chen J. Kirn D. McCormick F. Symons F. Nature. 1995; 374: 457-459Google Scholar). p85α interacts directly with Rac-1 (21Tolias K.F. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1995; 270: 17656-17659Google Scholar). Ras activates Rac-1 indirectly as a consequence of PI3K-mediated phosphorylation of membrane PIs (22Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Google Scholar). PI(3,4,5)P3 binds to the guanosine nucleotide-exchange factor SOS, stimulating SOS to load Rac-1 with GTP, an essential event in Rac-1 activation. Rac-1, in turn, activates downstream signaling through PAK-1 and its mediators, which include mitogen-activated protein kinase kinase-4 (MKK4) and its substrates c-Jun NH2-terminal kinase (JNK) and p38/HOG1 (23Davis R.J. Cell. 2000; 103: 239-252Google Scholar). Certain cancer cell types with PTEN gene loss have constitutively active PI3K and undergo apoptosis in response to pharmacologic or genetic inhibition of PI3K (24Neshat M.S. Mellinghoff I.K. Tran C. Stiles B. Thomas G. Petersen R. Frost P. Gibbons J.J. Wu H. Sawyers C.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10314-10319Google Scholar). Most non-small cell lung cancer (NSCLC) cell lines demonstrate hallmarks of PI3K pathway activation, such as phosphorylation of AKT and its downstream mediators, but have a wild-type PTEN gene (25Brognard J. Clark A.S. Ni Y. Dennis P.A. Cancer Res. 2001; 61: 3986-3997Google Scholar, 26Massion P. Kuo W.L. Stokoe D. Olshen A.B. Treseler P.A. Chin K. Chen C. Polikoff D. Jain A.N. Pinkel D. Albertson D.G. Jablons D.M. Gray J.W. Cancer Res. 2002; 62: 3636-3640Google Scholar, 27Forgacs E. Biesterveld E.J. Sekido Y. Fong K. Muneer S. Wistuba I.I. Milchgrub S. Brezinschek R. Virmani A. Gazdar A.F. Minna J.D. Oncogene. 1998; 17: 1557-1565Google Scholar, 28Moore S.M. Rintoul R.C. Walker T.R. Chilvers E.R. Haslett C. Sethi T. Cancer Res. 1998; 58: 5239-5247Google Scholar, 29Yokomizo A. Tindall D.J. Drabkin H. Gemmill H. Franklin W.A. Yang P. Sugio K. Smith D.I. Liu D. Oncogene. 1998; 17: 475-479Google Scholar, 30Soria J.C. Lee H.Y. Lee J.I. Wang L. Issa J.P. Kemp B.L. Liu D.D. Kurie J.M. Khuri F.R. Clin. Cancer Res. 2002; 8: 1178-1184Google Scholar). Despite having wild-type PTEN, NSCLC cells undergo apoptosis in response to PI3K pathway inhibition (25Brognard J. Clark A.S. Ni Y. Dennis P.A. Cancer Res. 2001; 61: 3986-3997Google Scholar). The apoptosis reported by Brognard et al. (25Brognard J. Clark A.S. Ni Y. Dennis P.A. Cancer Res. 2001; 61: 3986-3997Google Scholar) may depend in part on the absence of serum, which rescues cells from apoptosis induced by PI3K inhibition (18Li D.M. Sun H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15406Google Scholar, 19Lu Y. Lin Y.Z. LaPushin R. Cuevas B. Fang X. Yu S.X. Davies M.A. Khan H. Furui T. Mao M. Zinner R. Hung M.C. Steck P. Siminovitch K. Mills G.B. Oncogene. 1999; 18: 7034-7045Google Scholar, 31Lin J. Adam R.M. Santiestevan E. Freeman M.R. Cancer Res. 1999; 59: 2891-2897Google Scholar, 32Furnari F.B. Huang H.J. Cavenee W.K. Cancer Res. 1998; 58: 5002-5008Google Scholar). Thus, serum-induced activation of other peptide growth factor-induced signaling pathways can overcome the pro-apoptotic effect of PI3K inhibition. In this study, we investigated the signaling pathways that interact with PI3K to control NSCLC cell survival. Using pharmacologic and genetic approaches, we found that inhibition of PI3K-dependent signaling alone induced proliferative arrest, whereas inhibition of both PI3K and MKK4/JNK-dependent pathways induced apoptosis. These findings indicate that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival. Reagents—H358, H661, Calu-6, H460, H226B, H226Br, H441, and H1299 NSCLC cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum (complete medium). COS-7 cells and MKK4-null and wild-type mouse embryo fibroblast (MEF) cells (33Nishina H. Fischer K.D. Radvani L. Shahinian A. Hakem R. Rubie E.A. Bernstrin A. Mak T.W. Woodgett J.R. Penninger J.M. Nature. 1997; 385: 350-353Google Scholar) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. We purchased epidermal growth factor (EGF) (Invitrogen), insulin-like growth factor-1 (IGF-1) (R&D Systems, Minneapolis, MN), the class I PI3K inhibitor LY294002 (Calbiochem, La Jolla, CA), tumor necrosis factor-α (Sigma), recombinant GST-c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA), myelin basic protein (MBP) (New England Biolabs, Beverly, MA), GST-GSK3β (Santa Cruz), and protein A-G-agarose beads (Santa Cruz). We also purchased rabbit polyclonal antibodies against human phospho-AKT (pAKT1; Ser473) and AKT1 (New England Biolabs), phospho-JNK (Thr183/Tyr185; Cell Signaling Technologies), p85α, cyclin-dependent kinase (CDK) 2, and p27 (Santa Cruz), and murine monoclonal antibodies against human PTEN (Santa Cruz), phosphoextracellular signal-regulated kinase (ERK) (Thr202/Tyr204; Cell Signaling), caspase-3 and -9 (BD Pharmingen), poly(ADP-ribose) polymerase (VIC5) (Roche Diagnostics), and goat polyclonal antibodies against human ERK1/2, JNK-1, and β-actin (Santa Cruz). The adenoviral vector expressing wild-type p85α (Adex1CAp85α-HA) has been described elsewhere (34Ueki K. Algenstaedt P. Mauvais-Jarvis F. Kahn C.R. Mol. Cell. Biol. 2000; 20: 8035-8046Google Scholar). A recombinant adenovirus expressing human PTEN under the control of a cytomegalovirus (CMV) promoter was a gift from Dr. W. K. A. Yung (M. D. Anderson Cancer Center). Plasmid expression vectors containing Rac-1 (Val12) and MKK1 (R4F) were gifts from Dr. Melanie Cobb (The University of Texas Southwestern Medical Center, Dallas, TX). Generation of Ad5-Δp85—Δp85 is a bovine p85α mutant lacking 35 amino acids (residues Met479 to Lys513) in the inter-SH2 region that are necessary for binding to the p110 catalytic subunit (35Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Google Scholar). The Δp85 cDNA was inserted into the 5′ end of the bovine growth hormone polyadenylation signal at the HindIII site of the pAd-shuttle vector, which was a gift from Dr. Jack Roth (M. D. Anderson Cancer Center). The Δp85-containing shuttle vector was digested with BstI/ClaI and inserted into the pAd-speed vector (36Ji L. Nishizaki M. Gao B. Burbee D. Toyooka S. Kamibayashi C. Xu K. Yen N. Atkinson E.N. Fang B. Lerman M.I. Roth J.A. Minna J.D. Cancer Res. 2002; 62: 2715-2720Google Scholar). 293 cells were transfected with the resulting plasmid and then maintained until the onset of the cytopathic effect. Viral titers were determined by plaque assays and spectrophotometric analysis. The presence of Δp85 in viral particles was confirmed by dideoxy-DNA sequencing and Western blot analysis. Cell Growth Assays—NSCLC cell lines were seeded at 1–2 × 103 cells/well in 96-well plates. After 24 h, cells were incubated in serum-free conditions with 5 × 102, 1 × 103, 5 × 103, or 1 × 104 p/cell of Ad5-Δp85, Ad5-PTEN, or Ad5-CMV (control virus). After 2 h, cells were changed to complete medium. In the case of LY294002 treatment, cells were treated with 0.2, 2, 20, 40, 60, or 80 μm LY294002 in complete medium, which was changed every 48 h. After 5 days, cell growth was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Western Blot Analysis—Whole cell lysates were prepared by incubating cell pellets in lysis buffer (50 mm HEPES (pH 7.5), 150 mm NaCl, 1.5 mm MgCl2, 1 mm EDTA, 0.2 mm EGTA, 1% Nonidet P-40, 10% glycerol, 1mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 20 mm sodium fluoride, 5 mm sodium orthovanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin, and 1 mm benzamidine) for 20 min on ice. After clarification by centrifugation at 13,000 × g for 20 min, the supernatants were collected, and the protein concentration was determined with a BCA protein assay kit (Pierce). Cell lysates (30 μg) were subjected to SDS-PAGE and transferred onto a polyvinylidene fluoride nitrocellulose membrane (Bio-Rad). Membranes were immunoblotted overnight at 4 °C with primary antibodies in Tris-buffered saline containing 5% nonfat dry milk. Antibody binding was detected with an electrochemiluminescence kit (Amersham Biosciences) according to the manufacturer's directions. Cell Cycle and Apoptosis Assays—For these experiments, 1 × 106 H1299 cells were transferred onto 100-mm plates. Twenty-four hours later, the cells were incubated with 1 × 103,5 × 103,or1 × 104 particles of Ad5-Δp85 or Ad5-PTEN per cell. For combination treatments, H1299 cells were transiently transfected with 5 μg of plasmids containing Rac-1 (Val12), MKK1 (R4F), or empty vector using FuGENE (Roche Diagnostics). After 6 h, the cells were incubated for 2 h in serum-free conditions with Ad5-Δp85 or Ad5-CMV at 1 × 103 or 5 × 103 particles/cell. Cells were allowed to grow in complete medium for 48 h before being subjected to apoptosis assays. Apoptosis and cell cycle progression were measured by TUNEL with the APO-BRDU staining kit (Phoenix Flow Systems, San Diego, CA). Floating cells and attached cells were dispersed with trypsin-EDTA, pelleted, washed, and fixed in 1% paraformaldehyde for 15 min on ice and then fixed in 70% ethanol. The fixed cells were washed and incubated with DNA labeling solution containing terminal deoxynucleotidyltransferase reaction buffer, deoxynucleotidyltransferase enzyme, and bromodeoxyuridine triphosphate (BrdUrd-dUTP). The cells were rinsed before being resuspended with fluorescein-PRB-1 antibody solution and analyzed by flow cytometry in the presence of propidium iodide/RNase solution. Analyses of 3,000 to 10,000 events were done with a FACScan flow cytometer (BD Pharmingen) equipped with a 488-nm argon ion laser and two software packages: CellQuest 3.1 (BD Pharmingen) and ModFit LT 2.0 (Verity Software House, Topsham, ME). Live gating of the forward and orthogonal scatter channels was used to exclude debris and to selectively acquire cell events. A dual display of DNA area (linear red fluorescence) and BrdUrd-dUTP incorporation (FITC-PRB-1) was used to determine the percentage of propidium iodine-stained cells that were apoptotic. Apoptosis was also determined by the detection of nucleosomal DNA fragmentation by using the TACS apoptotic DNA laddering kit (Trevigen, Inc., Gaithersburg, MD) according to the manufacturer's protocol. Briefly, DNA was isolated from cells after adenovirus transfection or LY294002 treatment by incubating them in lysis buffer. DNA samples were subjected to electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining. Immune Complex Kinase Assay—H1299 cells were incubated for 2 h with Ad5-CMV, Ad5-Δp85, or Ad5-PTEN at 1 × 103, 5 × 103, or 1 × 104 p/cell in serum-free conditions, changed to complete medium, and incubated for 48 h. Cells were then washed twice in 1× phosphate-buffered saline, serum-starved for 24 h, treated with 50 ng/ml EGF for 15 min, and lysed in lysis buffer. Extracts were subjected to immunoprecipitation (100 μg) with antibodies to JNK1, AKT1/2, or ERK1/2 by rotation at 4 °C overnight. Protein A-G-agarose beads (20 μl) were added, and the solution was incubated at 4 °C for 1 h. The beads were washed three times with lysis buffer and once with kinase buffer (20 mm Hepes (pH 7.5), 20 mm β-glycerol phosphate, 10 mm MgCl2, 1 mm dithiothreitol, and 50 mm sodium orthovanadate). Kinase assays were performed by incubating the beads with 30 μl of kinase buffer, to which 20 μm cold ATP, 5 μCi of [γ-32P]ATP (2,000 cpm/pmol), and 2 μg of GST-c-Jun, GST-GSK3β, or MBP as substrates were added. The kinase reaction was performed at 30 °C for 20 min. The samples were then suspended in 1× Laemmli buffer and boiled for 5 min, and the samples were analyzed by 12% SDS-PAGE. The gel was dried and autoradiographed. Immune complex assays were also performed with COS-7 cells, which were transiently transfected for 6 h with 5 μg of plasmids containing Rac-1 (Val12), MKK1 (R4F), or empty vector using FuGENE. The cells were then transfected with Ad5-Δp85 or Ad5-CMV (1 × 103 or 5 × 103 particles/cell) and incubated in complete medium for 24 h. The cells were then changed to serum-free medium for 24 h, treated with IGF-1 (50 ng/ml) for 15 min, and lysed. JNK and ERK were immunoprecipitated from 100 μg of total cell lysates and subjected to kinase assays using GST-c-Jun and MBP, respectively, as substrates. Rac-1 Activity Assays—Pull-down assays with GST-tagged p21 binding domain (PBD) of PAK-1 were performed as follows. COS-7 cells were co-transfected with 2 μg of HA-tagged p85α, HA-tagged p110α, and Δp85 (2, 4, or 6 μg) using LipofectAMINE (Invitrogen). Total amount of DNA transfected per plate was equalized with empty vector. After 6 h, transfectants were washed and changed to normal growth medium. After 24 h, transfectants were serum-starved for 16 h, treated with 50 ng/ml EGF or IGF-1 for 15 min, and lysed. PAK-1 PBD-agarose (5 μg in a 50% slurry) was added to the lysates and the mixture was incubated for 1 h at 4 °C. The bead pellet was collected by centrifugation (5 s at 14,000 × g) and the supernatant was drained off. The beads were then washed and suspended in 20 μl of 1× Laemmli sample buffer. Proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose membrane, and blotted against Rac-1 and CDC42 polyclonal antibodies. PI3K-dependent Pathway Contributes to NSCLC Cell Proliferation and Survival—We investigated the effects of PI3K inhibition on the proliferation and viability of H1299 NSCLC cells, which have a wild-type PTEN gene (27Forgacs E. Biesterveld E.J. Sekido Y. Fong K. Muneer S. Wistuba I.I. Milchgrub S. Brezinschek R. Virmani A. Gazdar A.F. Minna J.D. Oncogene. 1998; 17: 1557-1565Google Scholar). H1299 cells were transfected with recombinant adenoviruses that express PTEN (Ad5-PTEN) or Δp85 (Ad5-Δp85), a p85α dominant-negative mutant lacking the inter-SH2 residues required for binding to the p110 catalytic domain (35Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Google Scholar). Transfection of H1299 cells with Ad5-PTEN or Ad5-Δp85 increased the expression of the adenoviral gene products and suppressed pAKT levels (Fig. 1), providing evidence that these adenoviral vectors effectively blocked PI3K-dependent signaling. When H1299 cells were incubated with Ad5-PTEN or Ad5-Δp85, cell number decreased in a dose-dependent fashion (Fig. 2, A and B). H1299 cell number also decreased in a dose-dependent manner after treatment with LY294002, a competitive inhibitor of ATP binding to all isoforms of class I PI3K (Fig. 2C). Other NSCLC cell lines with wild-type PTEN (H358, Calu-6, H460, H661, H226B, H441, H1299, and H226Br) underwent a similar decrease in cell number following treatment with LY294002 or transfection with Ad5-Δp85 or Ad5-PTEN (data not shown).Fig. 2Effect of PI3K inhibition on H1299 cell numbers. H1299 cells were (A) incubated with the indicated titers of Ad5-PTEN or Ad5-CMV, (B) incubated with the indicated titers of Ad5-Δp85 or Ad5-CMV, or (C) treated with medium alone (0) or the indicated doses of LY294002. The cells were incubated for 5 days, at which time they were subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Results are expressed relative to the density of cells treated with medium alone. Each value is the mean (± S.D.) of five identical wells.View Large Image Figure ViewerDownload (PPT) We next investigated whether PI3K inhibition induced proliferative arrest or apoptosis of NSCLC cells by performing flow cytometric analysis of H1299 cells transfected with Ad5-PTEN or Ad5-Δp85 and then stained with propidium iodide (Fig. 3A). Ad5-PTEN transfection induced proliferative arrest in the G0/G1 phase of the cell cycle, with minimal evidence of programmed cell death, as shown by the lack of a hypodiploid peak. Although Ad5-Δp85 transfection also caused an accumulation of cells in G1, its most striking effect was apoptosis, as indicated by the appearance of a hypodiploid peak. We examined this finding further by using terminal deoxynucleotidetransferase nick-end labeling (TUNEL), a more sensitive assay for apoptosis, and found low levels of DNA fragmentation in cells transfected with Ad5-PTEN (Fig. 3B). In contrast, transfection with Ad5-Δp85 produced much more DNA fragmentation, which is compatible with the induction of high levels of apoptosis (Fig. 3B). We investigated the effect of Ad5-PTEN and Ad5-Δp85 on signaling events known to contribute to apoptosis, proliferative arrest, or both (Fig. 4). Ad5-Δp85 transfection reduced the levels of procaspase-9, procaspase-3, and poly(ADP-ribose) polymerase, demonstrating evidence of caspase activation and proteolysis of a caspase-3 substrate. In contrast, Ad5-PTEN transfection decreased CDK2 levels and increased p27 CDK inhibitor levels without evidence of caspase activation or poly-(ADP-ribose) polymerase cleavage. Together, these findings support a role for PI3K in the proliferation of NSCLC cells and demonstrate a pro-apoptotic effect of Δp85. Δp85 Inhibits the Activity of MAP Kinases—Δp85 induced apoptosis of NSCLC cells whereas PTEN did not. Therefore, we hypothesized that inhibition of the PI3K/AKT pathway was required but not sufficient to induce apoptosis. We sought to identify additional survival signals typically activated by peptide growth factors that are inhibited by Δp85. Receptor tyrosine kinases maintain NSCLC cell survival, in part, by activating MAP kinases (37Schlessinger J. Cell. 2000; 103: 211-225Google Scholar). We investigated the role of MAP kinases in Δp85-induced cell death. H1299 NSCLC cells were incubated with Ad5-PTEN or Ad5-Δp85, treated with EGF, and subjected to in vitro kinase assays of JNK and ERK activity (Fig. 5). ERK activity increased in cells incubated with Ad5-CMV. Ad5-PTEN and Ad5-Δp85 had similar, dose-dependent effects on ERK activity. Relative to the effect of Ad5CMV, ERK activity increased with low dose (103 particles/cell) and decreased with high dose (5 × 103 or 104 particles/cell) Ad5-PTEN or Ad5-Δp85. JNK activity decreased minimally after Ad5-PTEN and, to a much greater extent, after Ad5-Δp85 incubation. Thus, Δp85 was unique in its ability to inhibit JNK activity. We investigated the mechanism by which Δp85 inhibited JNK. p85α associates with Rac-1, an upstream activator of JNK, and activates Rac-1 through association with a multiprotein complex that binds to p85 SH2 domains (38Innocenti M. Fritolli E. Ponzanelli I. Falck J.R. Brachman S.M. Di Fiore P.P. Scita G. J. Cell Biol. 2003; 160: 17-23Google Scholar). We investigated whether wild-type p85α and Δp85 differ in their ability to activate Rac-1. We quantitated Rac-1 activity in cell extracts using a pull-down assay with a GST-tagged PBD of PAK-1, which associates selectively with GTP-bound (activated) Rac-1 or CDC42. PBD-associated proteins are subjected to Western analysis to quantitate Rac-1 and CDC42. We performed this experiment in COS-7 cells, in which peptide growth factors activate Rac-1 through a PI3K-dependent mechanism (21Tolias K.F. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1995; 270: 17656-17659Google Scholar, 22Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Google Scholar). Using this assay we showed that Rac-1 is activated by treatment with EGF or IGF-1 (Fig. 6A). COS cells were co-transfected with wild-type p85 and increasing amounts of Δp85 and treated with EGF to activate Rac-1. Relative to the effect of wild-type p85, Δp85 inhibited peptide growth factor-induced activation of Rac-1 but not CDC42 (Fig. 6B). Thus, in contrast to the stimulatory effect of p85α, Δp85 inhibited Rac-1. p85α serves both to stabilize p85 protein and to inactivate PI3K lipid kinase activity (39Cuevas B.D. Lu Y. Mao M. Zhang J. LaPushin R. Siminovitch K. Mills G.B. J. Biol. Chem. 2001; 276: 27455-27461Google Scholar). Therefore, we tested the hypothesis that Ad5-Δp85 inhibits intracellular signaling activity by increasing intracellular p85 protein levels. We incubated H1299 NSCLC cells with various doses of Ad5-Δp85 or an adenoviral vector expressing full-length p85α (Adex1CAp85α-HA) and examined their rel" @default.
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• W2166608001 title "Evidence That Phosphatidylinositol 3-Kinase- and Mitogen-activated Protein Kinase Kinase-4/c-Jun NH2-terminal Kinase-dependent Pathways Cooperate to Maintain Lung Cancer Cell Survival" @default.
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