Requirement for the PI3K/Akt pathway in MEK1-mediated ... - Nature

Leukemia (2003) 17, 1058–1067 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu

MOLECULAR TARGETS FOR THERAPY (MTT)

Requirement for the PI3K/Akt pathway in MEK1-mediated growth and prevention of apoptosis: identification of an Achilles heel in leukemia WL Blalock1,5, PM Navolanic1, LS Steelman1, JG Shelton1, PW Moye1, JT Lee1, RA Franklin1,2, A Mirza3, M McMahon3, MK White4 and JA McCubrey1,2 1 Department 2

of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, NC, USA Leo Jenkins Cancer Center, Brody School of Medicine at East Carolina University, Greenville, NC, USA; 3UCSF/Mt. Zion Cancer Center, San Francisco, CA, USA; and 4Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson College of Medicine, Philadelphia, PA, USA

The Raf/MEK/ERK kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using DMEK1:ER, a conditionally active form of MEK1 which responds to either b-estradiol or the estrogen receptor antagonist 4 hydroxy-tamoxifen (4HT), we previously documented the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of human (TF-1) and murine (FDC-P1 and FL5.12) hematopoietic cells lines. Here we demonstrate the ability of DMEK1:ER to activate the phosphatidylinositol 3kinase (PI3K)/Akt/p70 ribosomal S6 kinase (p70S6K) pathway and the importance of this pathway in MEK1-mediated prevention of apoptosis. MEK1-responsive cells can be maintained long term in the presence of b-estradiol, 4HT or IL-3. Removal of hormone led to the rapid cessation of cell proliferation and the induction of apoptosis in a manner similar to cytokine deprivation of the parental cells. Stimulation of DMEK1:ER by 4HT resulted in ERK, PI3K, Akt and p70S6K activation. Treatment with PI3K, Akt and p70S6K inhibitors prevented MEK-responsive growth. Furthermore, the apoptotic effects of PI3K/Akt/p70S6K inhibitors could be enhanced by cotreatment with MEK inhibitors. Use of a PI3K inhibitor and a constitutively active form of Akt, [DAkt(Myr+)], indicated that activation of PI3K was necessary for MEK1-responsive growth and survival as activation of Akt alone was unable to compensate for the loss of PI3K activity. Cells transduced by MEK or MEK+Akt displayed different sensitivities to signal transduction inhibitors, which targeted these pathways. These results indicate a requirement for the activation of the PI3K pathway during MEK-mediated transformation of certain hematopoietic cells. These experiments provide important clues as to why the identification of mutant signaling pathways may be the Achilles heel of leukemic cell growth. Leukemia treatment targeting multiple signal transduction pathways may be more efficacious than therapy aimed at inhibiting a single pathway. Leukemia (2003) 17, 1058–1067. doi:10.1038/sj.leu.2402925 Keywords: MEK1; PI3K; Akt; P70S6K; signal transduction; oncogenes; cytokines

Correspondence: Dr JA McCubrey, Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Brody Building 5N98C, Greenville, NC 27858, USA; Fax: +1 252 744 3104 5

Current address: Shands Cancer Center, University of Florida, Gainesville, FL 32610, USA

Received 28 September 2002; accepted 7 February 2003

Introduction The proliferation and suppression of apoptosis in many hematopoietic precursor cells is promoted by interleukin-3 (IL3), granulocyte/macrophage-colony-stimulating factor (GM– CSF), stem cell factor, FL (the ligand for the tyrosine kinase receptors flt-2 and flt-3), thrombopoietin and certain other cytokines.1–6 Hematopoietic cell lines have been isolated which require IL-3 or in some cases GM-CSF for survival and cell proliferation.7 The murine FDC-P1 cell line is an IL-3/GM-CSFdependent cell line isolated from the bone marrow of a DBA/2 mouse.7 These cells represent early hematopoietic precursor cells with a colony-forming unit granulocyte/macrophage morphology. Cytokine deprivation of these cells results in rapid cessation of growth with subsequent death by apoptosis (programmed cell death).1–3,7–12 In the presence of IL-3 or GMCSF, these cells proliferate continuously; however, they are nontumorigenic when injected into immunocompromised mice.11 Spontaneous factor-independent cells are rarely recovered from the FDC-P1 cell line making it an attractive model to analyze the effects that various genes have on signal transduction and leukemogenesis, since abrogation of cytokine dependence is an important factor in the development of leukemia.1–3,8–12 IL-3 and GM-CSF exert their biological activity by binding to the IL-3 and GM-CSF receptors, respectively (IL-3R and GMCSFR).1,13–17 These receptors are comprised of a ligand-specific a-subunit and a common b-subunit (bc), which is essential for signal transduction.1,13–16 The membrane proximal domain of the bc chain is required for the induction of the Jak-STAT pathway, while the membrane distal domain is necessary for the stimulation of the Ras/Raf/MEK/ERK pathway.15 The activation of Jak2 by IL-3 may occur by receptor aggregation resulting in the multimerization of associated Jak2 proteins.14 Likewise, the binding of IL-3 to the receptor results in the formation of a complex, consisting of Grb2/Sos that is recruited to the bc chain by tyrosine phosphorylated Shc.18–23 The receptor-associated complex results in the stimulation of Ras, the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (PKB/Akt) pathway (hereafter referred to as Akt) and additional signal transduction molecules such as members of the Src family of tyrosine kinases.19 Ras promotes the sequential activation of Raf, MEK and the MAP kinases ERK1 and ERK2.22 This activation can further result

Interactions between MEK and PI3K/Akt/p70S6K in growth and apoptosis WL Blalock et al

1059 in the indirect or direct phosphorylation of transcription factors that regulate gene expression. PI3K can also be activated by Ras.20 Activated PI3K can result in the subsequent activation of the downstream targets phosphatidylinositol-dependent kinase1, Akt, p70 ribosomal S6 kinase (p70S6K) and NF-kB, many of which perform critical antiapoptotic functions.20–25 In this study, we report that aberrant activation of the Raf/MEK/ERK pathway can result in and requires the activation of the PI3K/Akt pathway. Akt family members all contain an amino-terminal pleckstrin homology (PH) domain, a catalytic domain and finally a carboxyl-terminal domain that lacks homology with other proteins. Activation of Akt occurs in two phases: (1) targeting to the lipid-rich cell membrane by the N-terminal PH domain and (2) phosphorylation on serine/threonine residues. The PH domain may mediate binding to certain lipid activators; however, the PH domain is not necessary for Akt kinase activity.26 Phosphorylation of T308 in the catalytic domain and S473 in the carboxyl-terminal domain results in the activation of Akt. Akt may serve to stimulate certain proteins involved in the prevention of apoptosis such as NF-kB as well as repress other proteins normally involved in the induction of apoptosis such as the forkhead transcription factors, glycogen synthetase-3 kinase, Fas, caspase 9 and cell cycle inhibitors such as p27Kip1.20 Activated Akt may phosphorylate certain downstream substrates such as IkK and stimulate NF-kB activity.20 As with other oncogenes, constitutively active forms of PI3K and Akt have been reported and certain spontaneous cytokine receptor mutants can result in constitutive activation of this pathway.26–29 Since activation of Akt by PI3K is dependent on localization of Akt to the plasma membrane, where it is subsequently phosphorylated, reported activating mutations of Akt include deletion of the N-terminal domain, which includes the PH domain.26 In these engineered deletion mutants, the Nterminus was substituted with the v-Src-encoded myristylation site so that Akt localization to the membrane was no longer dependent on PI3K activation. These mutants must still be phosphorylated in order to be active. Additionally, two key phosphorylation sites may be mutated to aspartic or glutamic acid residues to confer constitutive activation on Akt.26 Constitutive Akt activation has been associated with oncogenic transformation in NIH3T3 cells and the suppression of apoptosis in additional cell lines.26,30 Evidence suggests that the Ras/Raf/MEK/ERK pathway is intimately associated with the control of cell growth, differentiation and apoptotic machinery in myelo-monocytic cells.15,30–34 Recently, the downstream kinase ERK2 has been shown to phosphorylate the antiapoptotic Bcl-2 and the proapoptotic Bad molecules.35,36 Bcl-2 phosphorylation at serine 70 results in enhanced Bcl-2 antiapoptotic activity, whereas Bad phosphorylation results in its sequestering by 14-3-3 proteins.35–37 These modifications allow Bcl-2 to bind Bax and prevent apoptosis.20 In addition to phosphorylation by ERK, Bad may also be phosphorylated by Akt.20–23 Previous studies in FDC-P1 cells documented that a conditionally active form of MEK1, DMEK1:ER, could relieve the cytokine dependence of a subset of these cells.38–40 In the following study, the signal transduction events required for conditionally active MEK1 to relieve the cytokine dependence of FDC-P1 cells were further examined. The ability of MEK1 to activate the PI3K/Akt pathway and the requirement of PI3K activation in supporting MEK1-responsive growth and survival in FDC-P1 cells were determined. Understanding the roles the Raf/ MEK/ERK and the PI3K/Akt pathways play in the control of cell proliferation will increase our ability to suppress leukemia.

Materials and methods

Cell lines and growth factors Cells were maintained in a humidified 5% CO2 incubator. The IL-3/GM-CSF-dependent murine myeloid cell line FDC-P17 was cultured in RPMI 1640 (RPMI, Invitrogen, Carlsbad, CA, USA) medium supplemented with 10% WEHI-3B (D) conditioned medium as a source of murine IL-3, and 5% iron-supplemented bovine calf serum (BCS, Hyclone, Logan, UT, USA). MEK1dependent DMEK1:ER cells were grown in RPMI+5% FCS+1 mM b-estradiol or 500 nM 4-OH tamoxifen (4HT), an activator of the DMEK1:ER fusion protein38 (Sigma, St Louis, MO, USA). b-estradiol and 4HT were dissolved in ethanol. DMEK1: ER-infected cells were in some cases treated with the Raf inhibitor (5-iodo-3[(3,5-dibromo-4-hydroxyphenyl)methylene]2-indolinone, Calbiochem (San Diego, CA, USA), hereafter referred to as the Raf inhibitor), the MEK inhibitors, PD98059 (Cell Signaling, Beverly, MA, USA) or U0126 (Promega, Madison, WI, USA), the PI3K inhibitors, LY294002 and Wortmannin (Wort) (Calbiochem and Sigma), the Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol2-(R)-2-O-methyl-3-O-octadecylcarbonate, hereafter referred to as the Akt inhibitor, Calbiochem), the p70S6K inhibitor, rapamycin (Calbiochem) or the phorbol ester, phorbol 12-myristate 13-acetate (PMA, Sigma). These chemicals were dissolved in dimethyl sulfoxide (DMSO, Sigma). The optimal concentrations of the signal transduction inhibitors used were determined by titering these inhibitors on the cell lines and comparing which doses reduced [3H]thymidine and induced apoptosis as determined by annexin V/PI binding with the reported IC50s for these drugs. The specificity and mechanism of action of these inhibitors has been described.41

Retroviral infection of cells Cells were infected with a retrovirus (pBabepuro3)38–40 encoding a fusion protein (DMEK1:ER) between a constitutively activated form of MEK1 (DN3-S218E-S222D) and the hormone-binding domain of the human estrogen-receptor.38–40 This retrovirus also encodes resistance to the antibiotic puromycin (puror). In some cases, cells were also infected with a retrovirus containing a constitutively active form of Akt, DAkt(Myr+).42 The PH domain of Akt was deleted (DPH) and replaced with v-Src-derived myristolation site sequences (Myr+).42 This retrovirus contained a neomycin resistance gene neor for selection. Puror and neor FDC-P1 cells were isolated by selection in 1 mg/ml puromycin or 2 mg/ml G418 in the presence of IL-3, b-estradiol or 4HT as described.9 The nomenclature of the FDC-P1 cells is as follows: FD/DMEK1:ER for cells infected with the DMEK1:ER virus, FD/DMEK1:ER+DAkt(Myr+) for cells infected with DMEK1:ER and the DAkt(Myr+) retroviruses. (IL3) indicates that the cells were selected in IL-3, and (Est) or (4HT) indicates that the cells were selected in b-estradiol or 4HT, respectively.

Preparation of cell extracts and analysis by Western blotting Cells were washed twice with cold phosphate-buffered saline containing 5 mM Na2EDTA and lysed on ice in Gold lysis buffer as described.9 Cellular proteins were analyzed by electrophoresis through polyacrylamide gels followed by Western immunoblotting onto polyvinylidene difluoride (PVDF) membranes Leukemia


1060 (Immunobilon P; Millipore, New Bedford, MA, USA). Western blots were blocked in 1% bovine serum albumin and were then incubated with the indicated primary antibody [Santa Cruz Biotechnology (SCB), Santa Cruz, CA] at a dilution of 1:1000 as described.9 Phospho-specific ERK, Akt and p70S6K blots were conducted as above with the exception that the secondary antibody was an a-rabbit:HRP used at 1:2000 in TBST with 5% nonfat milk. The phospho-specific ERK, Akt and p70S6K Abs were purchased from Cell Signaling (Beverly, MA, USA). These protein samples were also run on parallel gels and probed with a nonphosphospecific Abs (Cell Signaling and SCB).

Determination of PI3K activity Protein lysates were prepared from cells cultured in phenol-redfree RPMI containing 5% charcoal-stripped FBS for 24 h. The cells were then stimulated with IL-3 or 4HT for 5 or 30 min. In some cases, the cells were pre-incubated with 10 mM Wort (Sigma) for 1 h before the addition of IL-3 or 4HT. The method of Auger et al,43 as modified by Zhang et al,44 was used to measure PI3K activity in immunoprecipitates. Cells were lysed in icecold lysis buffer (150 mM NaCl, 50 mM Tris HCl pH 7.4, 1% NP40, 1% sodium deoxycholate, 1 mM sodium ortho vanadate) with proteinase inhibitors (15 mg/ml aprotinin, 0.2 mM PMSF, 10 mg/ml leupeptin). A total of 100 mg of protein was diluted to a total volume of 500 ml for each sample with lysis buffer and 2 mg of rabbit polyclonal anti-mouse PI3K p85 subunit (SCB) added followed by an overnight incubation at 41C with mixing. Protein A–agarose (Boehringer-Mannheim GmbH, Germany) was added, followed by a further 2 h incubation with mixing at 41C. Immune complexes were precipitated at 2500 g for 5 min at 41C and washed twice in lysis buffer, without protease inhibitors, and twice in Tris-buffered saline EDTA (TBSE) with 1 mM ortho vanadate. TBSE contains 100 mM Tris pH 7.4, 150 mM NaCl and 1 mM EDTA. The final resuspension was in 50 ml TBSE. MnCl2 and phosphatidylinositol (Sigma) were added to final concentrations of 12.5 mM and 25 ng/ml, respectively. The reaction was started by the addition of 30 mCi of [g-32P]ATP (DuPont/NEN, Boston, MA, USA) and terminated after 10– 20 min at room temperature by the addition of 20 ml of 8 N HCl. A volume of 160 ml of CHCl3:CH3OH was added and the samples vortexed. The chloroform phase, which contains extracted lipids, was separated by centrifugation (10000 g, 5 min) and loaded on a silica gel thin layer chromatography (TLC) plate (Sigma). The TLC plates were developed in CHCl3:CH3OH:H2O:NH4OH (60:47:11.3:2), dried and visualized by autoradiography. Autoradiographs were scanned with a Hewlett-Packard HP1170Cse scanner and quantitated with the ImageQuant v3.3 software (Molecular Dynamics, Inc., Sunnyvale, CA, USA). The migration of phosphatidylinositol 30 phosphate was determined by running unlabeled phosphatidylinositol 30 -phosphate (Calbiochem) and staining with molybdenum blue (0.65% molybdenum oxide in 6 N H2SO4). The quantitation of the autoradiographs is shown with normalization to the untreated control.

Determination of Akt activity Cells were deprived of 4HT for 24 h in phenol-red-free RPMI medium that contained 5% charcoal-stripped BCS. The cells were then pulsed with cytokine or 4HT for varying periods of time. Akt activity was determined using an in vitro kinase assay. An aAkt1 Ab (SCB) and protein A–sepharose beads (Amersham) were used to immunoprecipitate the Akt1 protein overnight from Leukemia

100 mg of total protein lysate. The immunoprecipitates were washed once in GLB, once in GLB+500 mM NaCl once in Akt wash buffer (25 mM HEPES, pH 7.5 500 mM NaCl, 10% glycerol, 0.2 mM EGTA and 1% Triton X-100) and once in Akt reaction buffer (20 mM HEPES, pH 7.5, and 10 mM MgCl2). The immunoprecipitate was then used in a kinase reaction containing 20 mM HEPES pH 7.5, 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT, 200 mM ATP, 1 mM [g-32P]ATP and 1 mg histone 2B (H2B, Roche Diagnostics, Indianapolis, IN, USA) as a substrate. The reactions were carried out at 301C for 30 min before being stopped with 7.5 ml of sample buffer and boiling for 3 min. The reaction mixtures were electrophoresed through a 14% polyacrylamide gel, transferred to PVDF and exposed to a X-ray film. Western blots were preformed with 25 mg of the same samples with the aAkt1 Ab.

Determination of ERK activity Cells were deprived of 4HT for 24 h in phenol-red-free RPMI medium that contained 5% charcoal-stripped FCS. Then the cells were pulsed with cytokine or 4HT, for 15 min. ERK2 activity was determined after immunoprecipitation with an aERK2 antibody (SCB). The immunoprecipitated proteins were then used in a radioactive ERK assay as described.9,38–40

Caspase 3 activation and annexin V apoptosis assays Caspase 3 activity was measured using the APO LOGIXt carboxyfluorescein caspase detection kit (Cell Technology, Minneapolis, MN, USA). Approximately 1 107 exponentially growing cytokine-dependent, MEK1-responsive or MEK1/Aktresponsive cells were collected by centrifugation and annexin V/PI assays were performed as described.30,45 Controls, which were unstained, stained with annexin V only, or stained with PI only, were used to calibrate the FACS analyzer for each experiment.

Results

Effects of DMEK1:ER on cytokine dependence and downstream ERK activity We recently determined the effects of a conditionally active form of MEK1 on the cytokine dependence of hematopoietic cells.38–40 FDC-P1 cells were infected with a retrovirus encoding DMEK1:ER or an empty vector encoding only puromycin resistance (puror). While infection with the DMEK1: ER encoding retrovirus allowed some FDC-P1 cells to grow in medium lacking exogenous IL-3, no empty retroviral vectorinfected cells were recovered in medium containing puromycin and either b-estradiol or 4HT.38–40 The conditional activity of the DMEK1:ER protein was determined in FD/DMEK1:ER cells by treatment of 4HT-deprived cells with 4HT, IL-3, PMA or ethanol, and examining the presence of activated forms of downstream ERK by Western blotting (Figure 1a). 4HT was used in these studies as it is a more specific activator of DMEK1:ER than b-estradiol. Treatment of the cells with 4HT, IL-3 and PMA led to the detection of activated forms ERK (Figure 1a). Furthermore, treatment of the cells with 4HT, IL-3 and PMA led to the detection of ERK activity as determined by a kinase assay (Figure 1b and data not presented). Thus, these cells contain a conditional MEK1 gene, which can be activated by


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Figure 1 Effects of DMEK1:ER on ERK activity. MEK1-responsive FD/DMEK1:ER cells were deprived of 4HT for 24 h before being stimulated with IL-3, PMA, 4HT or ethanol for the indicated time periods. Western blot and ERK activities were determined with phospho-specific ERK Ab and an ERK assay as described in Materials and methods. The kinase assay was performed on protein lysates recovered from cells stimulated with IL-3 or 4HT for 15 min. As loading controls, Western and kinase blots were probed with an aERK2 Ab.

4HT and this results in downstream ERK activation. The following studies examine the ability of conditional MEK1 activity to result in the activation of the PI3K/Akt/p70S6K pathway, an essential pathway involved in regulation of cell cycle progression and the prevention of apoptosis.

Effects of inhibitors on DNA synthesis In order to determine the involvement of the PI3K/Akt/p70S6K pathway on MEK1-mediated proliferation, DNA synthesis was examined in the MEK-responsive cells treated with pharmacological inhibitors, which target the Raf/MEK/ERK and PI3K/Akt/ p70S6K pathways (Figure 2). In this figure, the data were plotted to maximize the visualization of the differences in the data, that is why the individual panels do not have the same scales on the Y-axis. The Raf inhibitor suppressed cytokine more than MEK1mediated DNA synthesis (panel A). The effects of this Raf inhibitor were examined as it has been speculated that MEK may induce Raf activity under certain circumstances.1 Since this inhibitor did not suppress MEK1-mediated DNA synthesis, it was not used in further studies. The MEK inhibitors (PD98059 and U0126) suppressed cytokine- and MEK-mediated DNA synthesis (panels B and C). MEK1-mediated DNA synthesis was more sensitive to the MEK inhibitors than cytokine-mediated DNA synthesis. Doses of 25 mM PD98059 and 10 mM U0126 were used in further studies based upon these and similar titration experiments using the induction of apoptosis as a readout (see Figure 6 and Table 1). The PI3K, Akt and p70S6K inhibitors suppressed MEK1, as well as, cytokine-mediated DNA synthesis (panels D, E and F). Doses of 10 mM LY294002, 25 mM Akt and 5 nM rapamycin were used in subsequent studies. In summary, data from [3H]thymidine incorporation assays indicated a strong requirement for functional PI3K, Akt and p70S6K activities for MEK1-mediated DNA synthesis.

Activation of MEK1 stimulates PI3K, Akt and p70S6K activities The effects of MEK1 activation on the PI3K pathway were examined (Figures 3–5). Treatment of MEK1-responsive cells with IL-3 and 4HT resulted in the detection of PI3K activity (Figure 3). IL-3 induced approximately four-fold higher levels of PI3K activity than untreated cells after 5 min (panel A). The level of PI3K activity decreased after 30 min of IL-3 treatment (panel B). Higher levels of PI3K activity were detected after 30 min (panel B) than 5 min (panel A) of 4HT treatment. The specificity of the induction of the PI3K activity was examined by the treatment of the cells with the PI3K inhibitor Wort. Wort was used in these experiments, as it is a more conventional and specific inhibitor of PI3K than LY294002.41 In these PI3K assays, the cells were only treated for 1 h, which avoids the problems associated with the lability of Wort.41 Wort pretreatment for 1 h suppressed the induction of PI3K treatment after IL-3 and 4HT addition (panels A and B). The effects of Wort on IL-3- and MEK1-mediated DNA synthesis were also examined. Wort suppressed both 4HT- and IL-3-mediated DNA synthesis (panel C). Thus, inhibition of PI3K activity by two different PI3K inhibitors, which act by different mechanisms, suppressed MEK1-responsive DNA synthesis. Activation of Akt was examined by two different techniques, which yielded similar results. Western blotting was performed with Akt activation-specific Abs (T308 and S473) (Figure 4, panel A). Akt kinase assay were performed with immunoprecipitated Akt protein and H2B as a substrate (Figure 4, panel B). After 5 min of 4HT treatment, induction of Akt activity was detected by both Western blotting and kinase assays. A similar time period of ERK activation was observed after 4HT treatment of these cells (Figure 1, panel A). As a control, the presence of activated forms of Akt was also examined after PMA treatment. PMA resulted in the prolonged detection of activated forms of Akt (Figure 4, panel A), as well as ERK (Figure 2, panel A). Leukemia


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Figure 2 Effects of kinase inhibitors on DNA synthesis. MEK1-responsive FD/DMEK1:ER cells treated with the signal transduction inhibitors were assayed for DNA synthesis by [3H]thymidine incorporation. The fold inhibition was calculated by dividing the level of DNA synthesis observed in the presence of DMSO and either IL-3 or 4HT by the level of DNA synthesis detected in the presence of the respective inhibitors and either IL-3 or 4HT. This experiment was preformed seven times, and the fold inhibitions were averaged together. In those cases where the standard deviation bars are not visible, they are contained within the size of the symbol. Panel (A) solid squares, IL-3- and Raf inhibitor-treated cells; solid triangles, 4HT- and Raf inhibitor-treated cells. Panel (B) solid squares, IL-3- and MEK inhibitor (PD98059)-treated cells; solid triangles, 4HT- and MEK inhibitor (PD98059)-treated cells. Panel (C) solid squares, IL-3- and MEK inhibitor (U0126)-treated cells; solid triangles, 4HT- and MEK inhibitor (U0126)-treated cells. Panel (D) solid squares, IL-3- and PI3K inhibitor (LY294002)-treated cells; solid triangles, 4HT- and PI3K inhibitor (LY294002)-treated cells. Panel (E) solid squares, IL-3- and AKT inhibitor-treated cells; solid triangles, 4HT- and Akt inhibitor-treated cells. Panel (F) solid squares, IL-3- and p70S6K inhibitor-treated cells; solid triangles, 4HT- and p70S6K inhibitor-treated cells.

Akt activity was detected within 5 min of IL-3 stimulation of MEK1-responsive cells. Akt activity dropped to lower levels approximately 1–2 h after IL-3 addition, but was detected at elevated levels 4–12 h after addition (Figure 4, panel B). MEK1 activation by the addition of 4HT resulted in the rapid activation of Akt (5–60 min) in MEK1-responsive FD/DMEK1:ER cells (Figure 4, panel B). Akt activity then dropped to near basal levels around 2 h and did not rise again until approximately 4 h of treatment. Thus, Akt activity displayed a biphasic response to both IL-3 and 4HT, as increases in Akt activity were detected early which were followed by decreases in activity, then finally elevated levels of Akt activity were detected after 4 h of treatment. p70S6K phosphorylation was examined in MEK1-responsive FD/DMEK1:ER cells using a phospho-specific antibody. p70S6K was phosphorylated rapidly in response to MEK activation (Figure 5). Thus, MEK activation in FD/DMEK1:ER cells results in the rapid activation of ERK, PI3K, Akt and p70S6K.

PI3K is required for MEK1-mediated growth In order to determine if the requirement of PI3K activity in MEK1-responsive proliferation could be by-passed by Akt, the Leukemia

induction of apoptosis was determined in MEK1 and MEK1/Aktresponsive cells. MEK1/Akt-responsive [FD/DMEK1:ER+DAkt(Myr+)] cells were derived by infecting cytokine-dependent FD/ DMEK1:ER cells, with a DAkt(Myr+) containing retrovirus and isolation of cells, which proliferated in the presence of 4HT and G418. Infection with an activated form of Akt increased the recovery of MEK1-responsive cells approximately 100-fold. Infection with activated Akt by itself was insufficient to abrogate the cytokine dependence of FDC-P1 cells, indicating the importance of the Raf/MEK/ERK pathway in controlling cytokine dependence. The effects of either MEK1 alone or MEK1+Akt on the induction of apoptosis were determined in the presence of inhibitors which targeted the PI3K/Akt/p70S6K or Raf/MEK/ERK pathways (Figure 6 and Table 1). Two independent assays for apoptosis were used in the following studies, detection of caspase 3 activation and annexin V/PI binding. Both of these techniques can be used to examine the induction of apoptosis.46–50 An example of these techniques is presented in Figure 6. Upon culture of the MEK1-responsive cells in the presence of 4HT, 27% of the cells registered as caspase 3 positive and 73% of the cells registered as caspase 3 negative (Figure 6, panel A). Of these cells, 65% registered as viable as determined by annexin V/PI binding (panel B, lower right quadrant, annexin V, PI negative), 19% of the cells registered as early apoptotic (panel


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Figure 3 Effects of MEK1 on PI3K activity. PI3K activity was examined in FD/DMEK1:ER cells that had been deprived of 4HT for 24 h and then subsequently stimulated with 4HT or IL-3 for 5 and 30 min (panels A and B, respectively). In some cases, the cells were pretreated with 10 mM Wortmannin (Wort) for 1 h before 4HT or IL-3 treatment. PI3K activity was determined by the incorporation of 32P from [g-32P]ATP into phosphatidylinositol. Phosphatidylinositol 30 phosphate was resolved by TLC as described in Materials and methods. PI3K activity was normalized to the levels detected in the unstimulated samples. Panels (A and B) PI3K activity after 5 and 30 min stimulation. Panel (C) Effects of Wort on DNA synthesis. The fold inhibition was calculated as described in the legend for Figure 2. These experiments were repeated three times and similar results were observed.

B, upper left quadrant, annexin V positive, PI negative), and 16% registered as necrotic/apoptotic (panel B, upper right quadrant, annexin V positive, PI positive). In Table 1, the annexin V-positive cells (upper left and upper right quadrants) have been added together. In this particular example, 27 and 35% of the cells registered as caspase 3 and annexin V positive, indicating that these two assays yielded similar results in terms of determining the extent of apoptosis. In contrast, when the

same cells were cultured in the presence of 4HT and the PI3K inhibitor, induction of apoptosis occurred as 83 and 90% of the cells registered as caspase 3 and annexin positive (panels C and D, respectively). The effects of PI3K pathway inhibitors on the induction of apoptosis on these and other MEK1- and Akt-infected cells were determined (Table 1). In these experiments, the effects of signal transduction inhibitors on three different types of DMEK1:ERinfected cells were examined, cytokine-dependent FD/DMEK1:ER(IL3), MEK1-responsive FD/DMEK1:ER(Est) and MEK1/Aktresponsive FD/DMEK1:ER+DAkt(Myr+)(4HT) cells. When the cells were cultured in IL-3 in the absence of any signal transduction inhibitor, the percentage of caspase 3-positive cells ranged from 19 to 34% and the percentage of annexin V-positive cells ranged from 13 to 34%. When the MEK1 and MEK1/Akt-responsive cells were cultured in 4HT, similar levels of caspase 3 and annexin-positive cells were observed. The cytokine-dependent cells were not cultured in the absence of IL-3 in these experiments as that resulted in approximately 100% caspase 3 and annexin V-positive cells (data not presented). When the cells were cultured in the presence of IL-3 and the PI3K inhibitor, the percentage of cells which registered as caspase 3 positive ranged from 17 to 47% and the percentage of cells which registered as annexin V positive ranged from 34 to 46% demonstrating the protective effects IL-3 has on apoptosis induced by the PI3K inhibitor. In contrast, when they were cultured in the presence of 4HT and the PI3K inhibitor, the percentage of cells, which registered as caspase 3 positive increased and ranged from 41 to 83% and the percentage of annexin V-positive cells ranged from 50 to 90%. The cells which grew in response to activated MEK1 alone (FD/ DMEK1:ER) were more sensitive to the effects of the PI3K inhibitor (83 and 90% caspase and annexin V positive, respectively) than the MEK/Akt-responsive cells, which overexpressed activated Akt [FD/DMEK1:ER+DAkt:ER(Myr+)(4HT)] (41 and 50% caspase 3 and annexin V positive, respectively) indicating that Akt overexpression could enhance viability when the cells were cultured with the PI3K inhibitor. Similar results were obtained when the cells were treated with either the Akt or p70S6K inhibitors. When the cells were cultured in the presence of IL-3, the percentage of caspase 3-positive cells ranged from 10 to 25% and the percentage of annexinpositive cells ranged from 20 to 48%. In contrast, when the cells were treated with 4HT and either the Akt or p70S6K inhibitors, the percentage of caspase 3-positive cells increased and ranged from 19 to 87% and the percentage of annexin-positive cells ranged from 33 to 94%. Again the cells which grew in response to activated MEK1 alone were more sensitive to the effects of the Akt and p70S6K inhibitors than the cells which overexpressed Akt indicating that Akt overexpression could enhance the viability of these cells when cultured with the Akt and p70S6K inhibitors. The effects of MEK inhibition on apoptosis were examined. When the cells were cultured in the presence of IL-3 and the MEK inhibitor, the percentage of caspase 3-positive cells ranged from 10 to 15% and the percentage of cells that registered as annexin V positive ranged from 18 to 30%. In contrast, when the cells were cultured in the presence of 4HT and the MEK inhibitor, the percentage of caspase 3-positive cell ranged from 43 to 80% and the percentage of annexin-positive cells ranged from 58 to 90%. The cells which grew in response to activated MEK1 alone, were more sensitive to the effects of the MEK inhibitor (80 and 90% caspase 3 and annexin positive, respectively) than the MEK/Akt-responsive cells, which activated Akt (43 and 58% caspase 3 and annexin positive, respectively), Leukemia


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Figure 4 Akt activation in FD/DMEK1:ER cells. MEK-responsive cells were deprived of 4HT for 24 h and then treated with IL-3, PMA, 4HT or ethanol for the indicated time periods. Panel (A) Western blots with phospho-specific Akt Abs and total Akt Ab as a control. Panel (B) Akt kinase assays. Akt1 was immunoprecipitated using an aAkt1 Ab. Immunoprecipitated Akt was used in an in vitro kinase assay with histone 2B (H2B) as a substrate. The Akt activity represents phosphorylated H2B. Aliquots of the cell lysates were electrophoresed on parallel gels and Western blotted with the aAkt1 Ab. These experiments were repeated twice and similar results were observed.

responsive cells were cultured in the presence of 4HT and the MEK and PI3K/Akt/p70S6K inhibitors, the percentage of caspase 3- and annexin-positive cells increased between 76 and 98% caspase and annexin positive indicating that inhibition of both pathways was an effective means to induce apoptosis.

Discussion Figure 5 DMEK1:ER induces p70S6K phosphorylation. MEK1responsive FD/DMEK1:ER cells were starved for 24 h and then treated with 4HT for the indicated times. Cells were lysed and the lysates electrophoresed on 10% SDS-PAGE gels. The gel was transferred to PVDF and immunoblotted with phospho-specific p70S6K Ab (1:1000) and an a-rabbit:HRP conjugate Ab. A parallel gel was Western blotted and probed with an Ab that recognizes total p70S6K (nonphosphospecific Ab).

indicating that Akt overexpression could enhance the viability of the cells when cultured with the MEK inhibitor. The effects of inhibition of both the MEK and PI3K/Akt/p70S6K pathways yielded more dramatic results. When the cytokinedependent FD/DMEK1:ER cells were cultured in the presence of IL-3 and MEK and PI3K inhibitors, approximately 85% of the cells registered as caspase 3 or annexin positive indicating that when both pathways were inhibited, apoptosis occurred even in the presence of IL-3. This effect was more dramatic when PI3K and MEK were inhibited as opposed to when more downstream components of the PI3K pathway were inhibited. Treatment of MEK1 or MEK1/Akt-responsive cells with MEK and PI3K, Akt, or p70S6K inhibitors in the presence of IL-3 yielded two distinct results. Upon culture with IL-3, the cells that expressed constitutively activated Akt were more sensitive to the induction of apoptosis with the combination of PI3K, Akt, p70S6K or MEK inhibitors. Of the cells, 41–72% registered as caspase 3 and annexin positive. These FD/DMEK1:ER+DAkt:ER(Myr+) cells were isolated on their ability to grow in response to MEK and Akt activation and were very sensitive to inhibition of the PI3K pathway even when cultured in the presence of exogenous IL-3. When the MEK1 and MEK1/AktLeukemia

Previously, we examined the ability of the dual-specific kinase MEK1 to transform a variety of hematopoietic cells to cytokine independence. Our studies revealed that aberrant MEK1 activity resulted in the conversion of a small subset of cells to cytokine independence.38–40 The transformation efficiency of MEK1 could be enhanced by the forced overexpression of the antiapoptotic protein Bcl-2, and we have since observed that elevated PI3K/Akt activity could also enhance the frequency of isolation of MEK1-responsive cells. In this study, we have examined some of the early effects of MEK1 activation in hematopoietic cells. Our results suggest that MEK1 may activate the PI3K/Akt/p70S6K pathway by MEK1 either directly or indirectly phosphorylating PI3K. Although beyond the scope of this paper, which focuses on the effects of these pathways on apoptosis and drug sensitivity, experiments are in progress to determine how MEK1 activates the PI3K pathway. Biphasic increases in Akt activity were observed in response to IL-3 and MEK activation. The initial activation of ERK, PI3K, Akt and p70S6K occurred within 5 min of 4HT treatment. This rapid activation was not observed after ethanol treatment and was not likely because of the de novo expression of autocrine growth factors. The second increase in Akt activity may result from the autocrine synthesis of growth factors, which serve to ‘restimulate’ the pathway. We have previously observed a biphasic induction of ERK activity in response to MEK1 activation in these cells.38–40 Of particular interest was the observation that inhibition of PI3K resulted in decreased long-term survival. PI3K is responsible for the phosphorylation of phosphatidylinositol converting it into a phosphatidylinositol 30 -phosphate second messenger


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Figure 6 Effects of signal transduction inhibitors on the induction of apoptosis. Cells were treated for 48 h with 4HT in medium containing DMSO (0.25%) (panels A and B) or PI3K inhibitor (LY294002, 10 mM) (panels C and D). Caspase 3 activity was measured by the Apo Logixt caspase 3 detection kit (panels A and C). Annexin V/PI binding were measured by the Roche kit (panels B and D). The lower left quadrant represents cells, which are negative for annexin V and PI (live cells). The upper left quadrant represents cells, which are positive for annexin V only (early apoptotic). The upper right quadrant represents cells, which are positive for both annexin V and PI (late apoptotic/necrotic). The lower right represents cells, which are positive for PI only (necrotic). At the corner of each quadrant is listed the percentage of cells in that particular quadrant. This experiment was performed three times and similar results were obtained.

Table 1

Effects of signal transduction inhibitors on the induction of apoptosisa FD/DMEK1:ER (IL3)

Growth conditions IL-3 4HT IL-3 4HT IL-3 4HT IL-3 4HT IL-3 4HT IL-3 4HT IL-3 4HT IL-3 4HT

FD/DMEK1:ER (Est)

FD/DMEK1:ER+Akt (Myr+)(4HT)

Inhibitor treatment

Caspase 3 positive (%)

Annexin V positive (%)





F F PI3K PI3K Akt Akt p70S6K p70S6K MEK MEK PI3K+MEK PI3K+MEK Akt+MEK Akt+MEK p70S6K+MEK p70S6K+MEK

31 F 47 F 20 F 19 F 10 F 85 F 59 F 34 F

20 F 46 F 18 F 20 F 19 F 86 F 59 F 36 F

19 27 17 83 25 79 10 87 10 80 34 95 20 95 17 94

34 35 35 90 48 88 32 94 18 90 46 98 32 97 29 91

18 18 19 41 22 19 15 34 15 43 60 90 41 76 61 78

13 14 34 50 27 33 29 48 30 58 74 92 62 85 72 94

a

The percentage of caspase 3-positive cells was determined as presented in Figure 6, The percentage of annexin V-positive cells represent the top left (annexin V positive, PI negative) and top right (annexin V positive, PI positive) quadrants of cells were determined as presented in Figure 6. F=Not done, Cytokine-dependent FD/DMEK1:ER cells were not cultured in the absence of IL-3 as greater than 75% of the cells would register as apoptotic in these conditions. Leukemia


1066 molecule responsible for the recruitment of various signaling molecules to the membrane where they are subsequently activated. One of these recruited molecules is Akt, which has been shown to phosphorylate Bad resulting in its sequestering by 14-3-3 protein family members, thus freeing Bcl-2 and resulting in enhanced cell survival. Another downstream consequence of the activation of PI3K is the activation of the p70S6K, which phosphorylates the S6 ribosomal protein as well as additional targets including Bad.51 In order to determine if Akt activation was the main role that PI3K was playing in MEK1-mediated transformation, FD/DMEK1:ER cells were infected with a constitutive DAkt(Myr+). Surprisingly, the combination of MEK1 and Akt was unable to suppress the requirement for PI3K, as double infectants were unable to survive in the presence of 4HT and the PI3K inhibitor. These data indicated that activation or recruitment of additional signal transduction molecules activated by PI3K, in addition to Akt, are required for MEK1responsive growth and survival. In general, FDC-P1 hematopoietic cells, which grew in response to MEK and Akt, displayed increased resistance to MEK and PI3K/Akt/p70S6K inhibitors when compared to cells, which just grew in response to activated MEK. However, when the MEK1/Akt-responsive cells were cultured in IL-3, they were more sensitive to cotreatment with MEK and PI3K/Akt/p70S6K pathway inhibitors. This may have therapeutic implications as these cells have revealed their Achilles heel, in that they are very sensitive to MEK and PI3K pathway inhibition. These cells should be useful in dissecting the interactions between signaling molecules, which result in the prevention of apoptosis. Moreover, these cells lines may prove useful in identifying which signal transduction pathways are better suited for targeted therapy. These studies also indicate that treatment of transformed hematopoietic cells with combinations of inhibitors may be a more effective method to inhibit leukemic growth than monotherapy. These experiments illustrate the importance of identifying the precise genetic lesions in leukemia, which may enable the physician to precisely treat the cancer patient with the correct cocktail of inhibitors. Recently, an important series of papers has been published in Leukemia which document the efficacy of treatment of leukemic cells with inhibitors, which target specific signal transduction pathways.53–61 Targeted cancer therapy provides promise to reduce the devastating toxic side effects of nonspecific chemotherapy.

Acknowledgements We thank Ms Catherine Spruill for the excellent artwork. This work was supported by grants from the United States National Institutes of Health (R01CA51025) and grants from the North Carolina Biotechnology Center (9805-ARG-0006, 2000-ARG0003) to JAM. RAF was supported in part by grants from the American Cancer Society (IRG-97-149), American Heart Association (9930099N) and the North Carolina Biotechnology Center (9705-ARG-0009). MKW was supported in part by Public Health Services Grants DK45718 from the National Institutes of Health.

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