Fibroblast Growth Factor-2 Induces Translational Regulation of Bcl-XL and Bcl-2 via a MEK-dependent Pathway
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m109006200
ISSN1083-351X
AutoresOlivier E. Pardo, Alexandre Arcaro, Giovanni Salerno, Selina Raguz, Julian Downward, Michael J. Seckl,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe involvement of fibroblast growth factor-2 (FGF-2) in the biology of small cell lung cancer (SCLC) has not previously been investigated. Here we report that FGF-2 prevented etoposide-induced apoptosis in H-510 SCLC cells. Phosphatidylinositol 3-kinase/protein kinase B signaling did not mediate this effect because FGF-2 failed to activate phosphatidylinositol 3-kinase or protein kinase B. In contrast, the mitogen-activated extracellularly regulated kinase kinase (MEK) was crucial for this response because its inhibition abolished the prosurvival properties of FGF-2. Moreover, in H-69 SCLC cells, the failure of FGF-2 to prevent etoposide-induced apoptosis correlated with uncoupling from MEK activation. However, the introduction of an activated MEK rendered these cells resistant to etoposide killing. Cell rescue relied on de novo protein synthesis, and the anti-apoptotic proteins Bcl-XL and Bcl-2 were up-regulated in a MEK-dependent fashion within 4 h of FGF-2 treatment. Contrary to previous reports, we found that this up-regulation occurred at the translational rather than the transcriptional level. Indeed, actinomycin D failed to prevent up-regulation of Bcl-XL and Bcl-2, and FGF-2 did not increase the mRNA levels or the stability of these proteins. The induction of the pro-apoptotic protein Bad by etoposide was also blocked by FGF-2 in a MEK-dependent fashion. Thus, MEK/extracellularly regulated kinase signaling is critical in the coordinate modulation of both pro- and anti-apoptotic Bcl-2 family members by FGF-2. The involvement of fibroblast growth factor-2 (FGF-2) in the biology of small cell lung cancer (SCLC) has not previously been investigated. Here we report that FGF-2 prevented etoposide-induced apoptosis in H-510 SCLC cells. Phosphatidylinositol 3-kinase/protein kinase B signaling did not mediate this effect because FGF-2 failed to activate phosphatidylinositol 3-kinase or protein kinase B. In contrast, the mitogen-activated extracellularly regulated kinase kinase (MEK) was crucial for this response because its inhibition abolished the prosurvival properties of FGF-2. Moreover, in H-69 SCLC cells, the failure of FGF-2 to prevent etoposide-induced apoptosis correlated with uncoupling from MEK activation. However, the introduction of an activated MEK rendered these cells resistant to etoposide killing. Cell rescue relied on de novo protein synthesis, and the anti-apoptotic proteins Bcl-XL and Bcl-2 were up-regulated in a MEK-dependent fashion within 4 h of FGF-2 treatment. Contrary to previous reports, we found that this up-regulation occurred at the translational rather than the transcriptional level. Indeed, actinomycin D failed to prevent up-regulation of Bcl-XL and Bcl-2, and FGF-2 did not increase the mRNA levels or the stability of these proteins. The induction of the pro-apoptotic protein Bad by etoposide was also blocked by FGF-2 in a MEK-dependent fashion. Thus, MEK/extracellularly regulated kinase signaling is critical in the coordinate modulation of both pro- and anti-apoptotic Bcl-2 family members by FGF-2. Fibroblast growth factor-2 induces translational regulation of Bcl-XL and Bcl-2 via a MEK-dependent pathway. CORRELATION WITH RESISTANCE TO ETOPOSIDE-INDUCED APOPTOSIS.Journal of Biological ChemistryVol. 290Issue 25PreviewVOLUME 277 (2002) PAGES 12040–12046 Full-Text PDF Open Access The development of tumor cell resistance to chemotherapy is the most frequent reason for failing to cure patients with common cancers such as small cell lung cancer (SCLC). 1The abbreviations used are: SCLCsmall cell lung cancerErkextracellularly regulated kinaseFGFfibroblast growth factorFGFRfibroblast growth factor receptorMEKmitogen-activated Erk kinasePI3Kphosphatidylinositol 3-kinasePKBprotein kinase BSFMserum-free mediumCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidS6KS6 kinaseFCSfetal calf serum Consequently, it is of central importance to elucidate the molecular mechanisms involved in drug resistance. Previous preclinical studies have implicated the overexpression of efflux proteins as a major mechanism of chemotherapy-induced resistance (1.Grant C.E. Valdimarsson G. Hipfner D.R. Almquist K.C. Cole S.P. Deeley R.G. Cancer Res. 1994; 54: 357-361PubMed Google Scholar). However, subsequent work using inhibitors of efflux proteins failed to demonstrate increased effectiveness of chemotherapy in patients (2.Ferry D.R. Traunecker H. Kerr D.J. Eur. J. Cancer. 1996; 32A: 1070-1081Abstract Full Text PDF PubMed Scopus (205) Google Scholar). This suggested the presence of alternative chemoresistance mechanisms. Indeed, other proteins involved in the ability of the cells to survive are elevated in tumors. In particular, levels of the anti-apoptotic protein Bcl-2 have been shown to correlate with the resistance of SCLC tumors to chemotherapy (3.Jiang S.X. Kameya T. Sato Y. Yanase N. Yoshimura H. Kodama T. Am. J. Pathol. 1996; 148: 837-846PubMed Google Scholar). Bcl-2 was originally identified in B-cell lymphomas (4.Tsujimoto Y. Finger L.R. Yunis J. Nowell P.C. Croce C.M. Science. 1984; 226: 1097-1099Crossref PubMed Scopus (1518) Google Scholar) and is now known to belong to a growing family of apoptosis regulatory proteins that may be either death antagonists (e.g. Bcl-2, Bcl-XL, and Bcl-w) or death agonists (e.g. Bax, Bad, Bak, Bcl-XS, and Bid) (5.Yang E. Korsmeyer S.J. Blood. 1996; 88: 386-401Crossref PubMed Google Scholar). The balance between these two types of Bcl-2 family members has been reported to partly control cell fate. Hence, overexpression of Bcl-2 (6.Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4452) Google Scholar) or Bcl-XL (7.Vander H. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1247) Google Scholar) has been shown to inhibit apoptosis, whereas overexpression of Bad (8.Jan M.S. Liu H.S. Lin Y.S. Biochem. Biophys. Res. Commun. 1999; 264: 724-729Crossref PubMed Scopus (24) Google Scholar) or Bax (9.Li X. Marani M. Yu J. Nan B. Roth J.A. Kagawa S. Fang B. Denner L. Marcelli M. Cancer Res. 2001; 61: 186-191PubMed Google Scholar) induces cell death. small cell lung cancer extracellularly regulated kinase fibroblast growth factor fibroblast growth factor receptor mitogen-activated Erk kinase phosphatidylinositol 3-kinase protein kinase B serum-free medium 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid S6 kinase fetal calf serum Fibroblast growth factor-2 (FGF-2) is a multifunctional cytokine involved in many biological processes including proliferation, differentiation, migration, neoangiogenesis, and cell survival (10.Friesel R.E. Maciag T. FASEB J. 1900; 9: 919-925Crossref Scopus (408) Google Scholar, 11.Rifkin D.B. Moscatelli D. J. Cell Biol. 1989; 109: 1-6Crossref PubMed Scopus (931) Google Scholar, 12.Song S. Wientjes M.G. Gan Y. Au J.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8658-8663Crossref PubMed Scopus (182) Google Scholar). Recent work has shown that fibroblast growth factors (FGFs), including FGF-2, can promote resistance to multiple chemotherapeutic agents both in vitro and in vivo (12.Song S. Wientjes M.G. Gan Y. Au J.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8658-8663Crossref PubMed Scopus (182) Google Scholar). This is of interest, because FGF-2 is frequently elevated in the serum of patients with various malignancies (13.Fujimoto K. Ichimori Y. Kakizoe T. Okajima E. Sakamoto H. Sugimura T. Terada M. Biochem. Biophys. Res. Commun. 1991; 180: 386-392Crossref PubMed Scopus (118) Google Scholar, 14.Linder C. Linder S. Munck W. Strander H. Anticancer Res. 1998; 18: 2063-2068PubMed Google Scholar). However, the molecular mechanisms by which FGF-2 promotes drug resistance in cancer cells have not been fully elucidated (15.Bryckaert M. Guillonneau X. Hecquet C. Courtois Y. Mascarelli F. Oncogene. 1999; 18: 7584-7593Crossref PubMed Scopus (57) Google Scholar). FGF-2 is known to bind and activate specific split tyrosine kinase receptors (FGFRs), which couple to multiple intracellular signaling pathways (16.Black E.G. Logan A. Davis J.R. Sheppard M.C. J. Endocrinol. 1990; 127: 39-46Crossref PubMed Scopus (56) Google Scholar, 17.Guimond S.E. Turnbull J.E. Curr. Biol. 1999; 9: 1343-1346Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The activation of tyrosine kinase receptors by other growth factors has been involved in cell survival through downstream signaling cascades such as the mitogen-activated Erk kinase (MEK)/extracellular regulated kinase (Erk) and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) pathways. These signals influence survival through several mechanisms including the regulation of Bcl-2 and its family members (15.Bryckaert M. Guillonneau X. Hecquet C. Courtois Y. Mascarelli F. Oncogene. 1999; 18: 7584-7593Crossref PubMed Scopus (57) Google Scholar, 18.Jost M. J. Biol. Chem. 2001; 276: 6320-6326Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 19.Liu Y.Z. Boxer L.M. Latchman D.S. Nucleic Acids Res. 1999; 27: 2086-2090Crossref PubMed Scopus (100) Google Scholar, 20.Pugazhenthi S. Nesterova A. Sable C. Heidenreich K.A. Boxer L.M. Heasley L.E. Reusch J.E. J. Biol. Chem. 2000; 275: 10761-10766Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar). This regulation may be rapid or delayed as a consequence of phosphorylation (21.Deng X. Ruvolo P. Carr B. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (225) Google Scholar) or increased transcription (18.Jost M. J. Biol. Chem. 2001; 276: 6320-6326Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), respectively. However, translational regulation of Bcl-2 family members in response to growth factors has not been described. In the present study we show that FGF-2 prevents apoptosis induced by the chemotherapeutic agent etoposide in SCLC cells. This effect occurs via a MEK-dependent and PI3K/PKB-independent pathway. We show that FGF-2 signaling through MEK increases the levels of the anti-apoptotic members of the Bcl-2 family, Bcl-2 and Bcl-XL, and blocks etoposide-mediated induction of the pro-apoptotic protein Bad. These effects were selective because Bax protein expression was unchanged. Surprisingly, the increase in Bcl-XL and Bcl-2 was not due to increased mRNA transcription but instead resulted from enhanced translation. This represents a new mechanism by which growth factor activation of MEK increases cell survival and resistance to chemotherapy. The H-510 and H-69 SCLC cell lines were maintained as previously described (22.Seufferlein T. Rozengurt E. Cancer Res. 1996; 56: 3895-3897PubMed Google Scholar). For experimental purposes, the cells were grown in serum-free medium (SFM; RPMI 1640 supplemented with 5 μg/ml insulin, 10 μg/ml transferrin, 30 nm sodium selenite, 0.25% bovine serum albumin) and used after 3–7 days. H-510 cells (5 × 104cells/ml SFM) were pretreated with or without various inhibitors: 25 μm PD098059, 20 μm U0126 (New England Biolabs), 10 ng/ml rapamycin, 10 μm LY294002 (Calbiochem), or 100 μm cycloheximide (Sigma). The cells were then stimulated with or without 0.1 ng/ml FGF-2 (Amersham Biosciences) for 4 h prior to treatment with 0.1 μmetoposide (Sigma) and incubated at 37 °C for 96 h. A single cell suspension was generated by passing the samples four times through a 19-G needle, and live cell number was determined using trypan blue exclusion. H-510 cells (2 × 106/5 ml SFM) were incubated with or without 0.1 ng/ml FGF-2 for 4 h prior to addition of 0.1 μm etoposide for 4 h. The cells were then resuspended at 1 × 106 cells/ml binding buffer (10 mm HEPES, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2). 100 μl of this suspension were mixed with 5 μl of fluorescein isothiocyanate-conjugated annexin V reagent (Molecular Probes) and 10 μl of propidium iodide solution (Molecular Probes). The samples were left for 15 min at room temperature in the dark before the addition of a further 400 μl of binding buffer. The samples were analyzed by flow cytometry using a Becton Dickinson FACS Vantage S.E. turbo. H-510 cells (2 × 106 cells/ml SFM) were incubated with or without 0.1 ng/ml FGF-2 for 4 h before the addition of 0.1 μmetoposide for 10 h. The cells were homogenized for 15 min at 4 °C in 1 ml of 10 mm Tris, pH 7.5, 10 mmNaH2PO4, Na2HPO4, 130 mm NaCl, 1% Triton X-100, 10 mm NaPPi in the absence of protease inhibitors. The lysates were centrifuged for 10 min at 15,000 × g, and 100 μl of supernatant was mixed with 100 μl of 50 mm HEPES, pH 7.4, 100 mmNaCl, 0.1% CHAPS, 10 mm dithiothreitol, 0.1 mmEDTA, 10% (v/v) glycerol. The proteolytic assay was initiated by the addition of 10 μl of 2 mm colorimetric substrate (Ac-DEVD-p-nitroaniline; Calbiochem) and was continued at 37 °C for 2 h. Caspase 3 activity was determined by measuring the absorbance at 405 nm. The cell lysates were subjected to SDS-PAGE and transferred to Immobilon-P membranes (Millipore) prior to probing with the following antibodies: anti-activated Erk1/2 (Sigma), anti-Bcl-2, Bcl-XL, Bad, poly(ADP ribose)polymerase, Lamin B, and β-actin (Santa Cruz), anti-Bax (Transduction Laboratories/Becton Dickinson). Monoclonal anti-p85α antibody (clone U2) was a generous gift from Dr I. Gout (Ludwig Institute, University College London, London, UK). Detection was performed using ECL (Amersham Biosciences). Optical density was determined using the LabWork software (UVP Laboratory Products). H-510 cells (1 × 107/ml RPMI) were stimulated with 10% fetal bovine serum or 0.1 ng/ml FGF-2 for 5 min at 37 °C. The samples were lysed for 20 min on ice in 2 ml of lysis buffer (20 mm HEPES-NaOH, pH 7.4, 150 mm NaCl, 1% (w/v) Triton X-100, 2 mmEDTA, 10 mm sodium fluoride, 10% (w/v) glycerol, 1 mm phenylmethylsulfonyl fluoride, 5 mmbenzamidine, 1 mm N-tosyl-l-lysine chloromethyl ketone, 20 μm leupeptin, 18 μm pepstatin, 20 μg/ml aprotinin, 1 mmdithiothreitol, 2 mm Na3VO4, 10 mm β-glycerophosphate). Immunoprecipitation was performed for 2 h at 4 °C with a p85α monoclonal antibody or an irrelevant mouse IgG. Protein G-Sepharose CL-4B (Amersham Biosciences) was then added, and the incubation was continued for 1 h at 4 °C. The immunoprecipitates were washed twice in lysis buffer and once in Tris-buffered saline (50 mm Tris-HCl, 150 mm NaCl, pH 7.4). PI3K activity was assayed by resuspending the immunoprecipitates in 25 μl of 2× kinase buffer (40 mm Tris-HCl, pH 7.4, 200 mm NaCl). Phosphoinositides were sonicated for 15 min in 50 mmTris-HCl, pH 7.4, and added to the samples (final concentration, 0.2 mg/ml). The kinase reactions were initiated in 50 μl of final volume by the addition of 3.5 mm of MgCl2, 40 μm ATP, and 10 μCi of [γ-32P]ATP (3000 Ci/mmol; Amersham Biosciences) and incubated for 10 min at 37 °C. The reactions were stopped by the addition of 100 μl of 1n HCl and 200 μl of CHCl3/MeOH (1:1) (v/v). The organic phase was collected, re-extracted with 40 μl of MeOH, 1n HCl (1:1) (v/v). The samples were then dried, resuspended in 30 μl of CHCl3/MeOH (1:1) (v/v), and spotted onto Silica Gel 60 TLC (Whatman) plates pretreated with 1% (w/v) oxalic acid, 1 mm EDTA, 40% MeOH (v/v) and baked for 15 min at 110 °C. The plates were developed in propanol, 2 macetic acid (65:35) (v/v), and the radiolabeled spots were detected by autoradiography. SCLC cells grown in SFM were washed three times in RPMI 1640, and 2 × 106 cell aliquots were incubated in this medium for 30 min at 37 °C. The cells were then stimulated with FGF-2 for 10 min and lysed at 4 °C in 1 ml of lysis buffer. Immunoprecipitation was performed for 2 h using a polyclonal anti-S6K1 antibody (Santa Cruz). Protein A-agarose beads (40 μl, 1:1 slurry) were added for a further 1 h. Immune complexes were washed twice in lysis buffer and three times in kinase buffer (20 mm HEPES, pH 7.4, 10 mm MgCl2, 1 mm dithiothreitol, 10 mm β-glycerophosphate). The kinase reaction was initiated by resuspending the beads in 25 μl of kinase buffer supplemented with 100 μm ATP, 100 μCi/ml [γ-32P]ATP, 200 μm microcystin LR, and 1 mg/ml S6 peptide (RRRLSSLRA). The reactions were incubated at 30 °C for 10 min (linear assay conditions) and terminated on ice by the addition of 10 μl of ice-cold 0.5% orthophosphoric acid. The samples were centrifuged (15,000 × g for 30 s), and 30 μl of the supernatant were spotted onto P81 chromatography paper (Whatman). The filters were washed four times for 5 min in 0.5% orthophosphoric acid, immersed in acetone, and dried before Cherenkov counting. The average radioactivity of two blank samples containing protein A-agarose beads (40 μl, 1:1 slurry) but no immune complex was subtracted from the result of each sample. The specific activity of the [γ-32P]ATP used was 900–1200 cpm/pmol. High titer murine retrovirus were obtained using a previously published method (23.Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2316) Google Scholar). SCLC cells grown in RPMI containing 10% FCS were subjected to primary retroviral infection using a human retrovirus coding for the ecotropic murine retrovirus receptor and bearing the neomycin resistance gene (24.Albritton L.M. Tseng L. Scadden D. Cunningham J.M. Cell. 1989; 57: 659-666Abstract Full Text PDF PubMed Scopus (586) Google Scholar). Briefly, 10 × 106 SCLC cells in 5 ml of RPMI containing 10% FCS were incubated overnight with 5 ml or retroviral preparation in Dulbecco's modified Eagle's medium containing 10% FCS and 8 μg/ml Polybrene (Sigma). The medium was then replaced with 20 ml of RPMI containing 10% FCS, and expression of the transgene was allowed to take place for 48 h. Infected cells were then selected in RPMI containing 10% FCS and 0.8 mg/ml G418. The secondary retroviral infection was then performed using a murine retrovirus vector bearing the puromycin resistance gene and coding for constitutively active MEK (S217E/S221E; kindly provided by Prof. Chris J. Marshall). After 48 h, selection was then performed in RPMI containing 10% FCS and 5 μg/ml puromycin for 7 days before cells were transferred to SFM for experimental use. H-510 cells were washed three times in Met-free SFM, aliquoted at 4 × 106 cells/ml of this medium and incubated for 1 h at 37 °C/5% CO2 with 150 μCi of [35S]Met/ml Promix (Amersham Biosciences) followed by a further 4 h with or without 0.1 ng/ml FGF-2. The cells were then washed three times in SFM and incubated with or without FGF-2 in the absence of Promix at 37 °C. The samples were lysed at 20-min intervals in 1 ml of lysis buffer and stored at −80 °C until the last time point was collected. Bcl-2 and Bcl-XL were immunoprecipitated for 3 h at 4 °C and analyzed by SDS-PAGE and autoradiography. The optical density of the bands was determined and plotted as a function of time. H-510 cells (4 × 106 cells/ml SFM) were incubated for 4 h with or without 0.1 ng/ml FGF-2. The total mRNA was extracted with RNAzol (Biogenesis), and reverse transcription was performed using the avian myeloblastosis virus first strand reverse transcription-PCR kit (Roche Molecular Biochemicals). Equal amounts of cDNA were then introduced in a TaqMan real time quantitative PCR. For Bcl-XL, primers with the sequences 5′-TCCTTGTCTACGCTTTCCACG-3′ and 5′-GGTCGCATTGTGGCCTTT-3′ were used together with a 5′-ACAGTGCCCCGCCGAAGGAGA-3′ Taqman probe. For Bcl-2, primers with the sequences 5′-CATGTGTGTGGAGAGCGTCAA-3′ and 5′-GCCGGTTCAGGTACTCAGTCA-3′ were used together with a 5′-CCTGGTGGACAACATCGCCCTGT-3′ Taqman probe. The probes were labeled at the 5′ end with the reporter 6-carboxy-fluorescein and at the 3′ end with the quencher molecule 6-carboxy-tetramethyl-rhodamine. Real time PCR amplification was performed according to the Taqman Universal PCR Master Mix protocol. Relative quantification of gene expression was obtained as described in the manual using glyceraldehyde-3-phosphate dehydrogenase mRNA as an internal standard. We have found that several SCLC cell lines including H-510 cells express FGFR1 and 2, which are known to bind FGF-2 with high affinity (25.Mansukhani A. Dell'Era P. Moscatelli D. Kornbluth S. Hanafusa H. Basilico C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3305-3309Crossref PubMed Scopus (116) Google Scholar, 26.Werner S. Duan D.R. Devries C. Peters K.G. Johnson D.E. William L.T. Mol. Cell. Biol. 1992; 12: 82-88Crossref PubMed Scopus (302) Google Scholar, 27.Ron D. Reich R. Chedid M. Lengel C. Cohen O.E. Chen A.M. Neufeld G. Miki T. Tronick S.R. J. Biol. Chem. 1993; 268: 5388-5394Abstract Full Text PDF PubMed Google Scholar, 28.Ornitz D.M. Xu J. Colvin J.S. McEwen D.G. MacArthur C.A. Coulier F. Gao G. Goldfarb M. J. Biol. 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The appearance of several apoptotic markers including annexin V staining, caspase 3 activation, and poly(ADP ribose)polymerase cleavage (Fig. 1, B–D) confirmed that apoptosis was induced by etoposide in H-510 cells. However, pretreatment with FGF-2 prevented the induction of these changes (Fig. 1, B–D). This was not a mere delay in the onset of the apoptotic process because FGF-2-treated cells did not show increased poly(ADP ribose)polymerase cleavage at time points as late as 35 h post-etoposide (data not shown). Our results therefore demonstrate that FGF-2 prevents etoposide-induced apoptosis in SCLC cells. PI3K/PKB signaling plays a key role in survival induced by other growth factors in many cell systems (30.Arase Y. Hiwasa T. Hasegawa R. Nomura J. Ito H. Suzuki N. Biochem. Biophys. Res. Commun. 2000; 267: 33-39Crossref PubMed Scopus (16) Google Scholar, 31.Minshall C. Arkins S. Dantzer R. Freund G.G. Kelley K.W. J. Immunol. 1999; 162: 4542-4549PubMed Google Scholar, 32.Suzuki A. Hayashida M. Kawano H. Sugimoto K. Nakano T. Shiraki K. Hepatology. 2000; 32: 796-802Crossref PubMed Scopus (104) Google Scholar). Moreover, in H-510 cells, we have found that S6K, which frequently lies downstream of PI3K, is activated in response to FGF-2 (see Fig. 4B). Therefore, we examined the effect of the PI3K inhibitor LY294002 on the ability of FGF-2 to prevent apoptosis in H-510 cells. When used at concentrations previously shown to block PI3K activity in SCLC cells (33.Moore S.M. Rintoul R.C. Walker T.R. Chilvers E.R. Haslett C. Sethi T. Cancer Res. 1998; 58: 5239-5247PubMed Google Scholar), the addition of LY294002 alone resulted in reduced cell survival to an extent similar to that seen with etoposide (Fig. 2A). Nevertheless, LY294002 did not prevent FGF-2 induced rescue of etoposide killing. Similarly, inactivation of S6K with rapamycin (22.Seufferlein T. Rozengurt E. Cancer Res. 1996; 56: 3895-3897PubMed Google Scholar) also failed to prevent FGF-2-induced rescue (Fig. 2A). Further evidence that PI3K was not involved in FGF-2-induced resistance to etoposide was provided by the failure of FGF-2 to stimulate PI3K in anti-p85α immunoprecipitates, although p85α was present, and PI3K activity could be induced by serum in H-510 SCLC cells (Fig. 2B). FGF-2 similarly failed to induce PI3K activity in anti-phosphotyrosine immunoprecipitates (data not shown). The activation of PKB relies on the phosphorylation of Thr-308 and Ser-473, and phospho-specific antibodies to the latter site have frequently been used as a simple and sensitive readout for any changes in PI3K activity. Strikingly, despite PKB being present in H-510 cells (Fig. 2C, upper panel), we were unable to detect Ser-473 phosphorylation in response to FGF-2 (Fig. 2C, lower panel). This was not a consequence of either the lack of sensitivity of the phospho-specific antibody or low protein loading, because samples from COS cells run in parallel showed inducible PKB phosphorylation despite lower total levels of this kinase. Taken together, these results strongly suggest that neither the PI3K/PKB pathway nor S6K mediate FGF-2-induced resistance to etoposide in H-510 cells.Figure 2The PI3K/PKB pathway is not involved in FGF-2 prosurvival activity. A, H-510 cells were pretreated with (+) or without (−) LY294002 (LY) or rapamycin (R) prior to preincubation with (+) or without (−) FGF-2. Etoposide (Eto) was then added, and cell survival was determined after 96 h. The results shown are the means ± S.E. of three independent experiments performed in quadruplicate. B, H-510 cells were incubated with or without FGF-2 or fetal bovine serum (FBS). PI3K activity was assessed in vitro on anti-p85α immunoprecipitates (upper panel). The amounts of p85α immunoprecipitated per condition were equivalent as shown by Western blot (lower panel). C, COS-1 and H-510 cells were treated with or without 100 ng/ml EGF or 0.1 ng/ml FGF-2, respectively. The levels of total (upper panel) or phospho-Ser-473-PKB (lower panel) were determined by Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Increasing evidence has implicated the MEK/Erk pathway as an alternative mechanism capable of providing cell survival signals (15.Bryckaert M. Guillonneau X. Hecquet C. Courtois Y. Mascarelli F. Oncogene. 1999; 18: 7584-7593Crossref PubMed Scopus (57) Google Scholar, 18.Jost M. J. Biol. Chem. 2001; 276: 6320-6326Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 19.Liu Y.Z. Boxer L.M. Latchman D.S. Nucleic Acids Res. 1999; 27: 2086-2090Crossref PubMed Scopus (100) Google Scholar, 21.Deng X. Ruvolo P. Carr B. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (225) Google Scholar, 34.Bonni A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Science. 1999; 286: 1358-1362Crossref PubMed Scopus (1697) Google Scholar). PD098059 is a well documented MEK1/2 inhibitor that has been extensively used to assess the role of MEK/Erk signaling in various biological processes (35.Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3262) Google Scholar). Dose response experiments indicated that addition of 25 μm of PD098059 was sufficient to maximally inhibit FGF-2-induced activation of Erk1/2 in H-510 cells (Fig. 3A). At this concentration, PD098059 induced only a modest reduction in SCLC cell survival. However, this inhibitor completely prevented FGF-2-induced rescue of H-510 cells from etoposide killing (Fig. 3B). To substantiate the notion that MEK could mediate FGF-2-induced resistance to etoposide, we performed similar experiments in the H-69 SCLC cell line. In these cells, FGF-2 failed to activate MEK/Erk signaling at all concentrations and times tested (Fig. 4A). This was not due to a dysfunctional MEK/Erk pathway because Erk phosphorylation could be induced using phorbol esters (Fig. 4A). Moreover, as in H-510 cells, FGF-2 was still able to induce other signaling events such as S6K activation (Fig. 4B). Nevertheless, the addition of FGF-2 failed to rescue etoposide-induced cell death in H-69 cells (Fig. 4C). Furthermore, introduction of an activated version of MEK into H-69 cells induced phosphorylation of Erk1/2 and complete rescue from etoposide killing (see Fig. 6). Taken together, these results indicate that MEK/Erk signaling is the principal mediator of FGF-2-induced rescue from etoposide killing in SCLC cells.Figure 6Constitutively activated MEK induces Bcl-XL and Bcl-2 expression and resistance to etoposide killing in H-69 cells. H-69 cells expressing EcoR with (+) or without (−) kinase active MEK (MEK-KA) were lysed and analyzed by immunoblotting for MEK, Bcl-XL, and Bcl-2 (A) and compared with FGF-2-treated H-510 cells for biphospho-Erk1/2 levels (B). Lamin B detection was used as a control for protein loading. C, H-69 cells expressing EcoR with kinase active MEK (MEK-KA) were treated with or without FGF-2 and compared with EcoR expressing H-69 cells for their ability to survive a 4-day treatment with 0.1 μm etoposide. In all cases, the data shown are representative of three independent experiments. For C the results are the means ± S.E. of quadruplicate samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Time course studies demonstrated that preincubation periods shorter than 3 h with FGF-2 were insufficient to rescue H-510 cells from etoposide killing. In contrast, the activation of Erk1/2 in response to FGF-2 was rapid and transient (peak, 5 min; control levels, 10 min) in these cells (data not shown). This discrepancy between transient Erk1/2 activation and the prolonged preincubation with FGF-2 necessary to prevent etoposide killing suggested the need for protein synthesis downstrea
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