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2 CRC Labs and Section of Cancer Cell Biology, Imperial College School of Medicine at Hammersmith Hospital, London W12 ONN, UK
3 Guy's, King's, St. Thomas's School of Medicine and Dentistry, The Rayne Institute, London SE5 9NU, UK
Address correspondence to Paul J. Coffer, Department of Pulmonary Diseases, G03.550, University Medical Center, Heidelberglaan 100, 3584 CX Utrecht, Netherlands. Tel.: 31-30-250-7134. Fax: 31-30-250-5414. E-mail: p.coffer{at}hli.azu.nl
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Abstract |
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These data demonstrate that activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal. Thus PI3K/PKBmediated inhibition of this transcription factor likely provides an important mechanism by which survival factors act to prevent programmed cell death.
Key Words: apoptosis; cytokine; mitochondria; PI3K; forkhead
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Introduction |
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A well-characterized mechanism of initiating apoptosis is through ligand-mediated activation of cell surface death receptors, such as the tumor necrosis factor receptors and CD95 (APO-1/Fas) (for review see Nagata, 1999). Caspase-8 is indispensable for transducing apoptotic signals initiated by death receptors, as demonstrated by the observation that CD95 signaling is abrogated in caspase-8-/- mice (Varfolomeev et al., 1998). Caspase activation can also be triggered via a death receptorindependent mechanism, involving the regulation of mitochondrial membrane permeability. Central to this "intrinsic" means of mitochondrial-initiated caspase activation is the release of cytochrome c from the intermembrane space of mitochondria into the cytosol. Cytochrome c, together with apoptosis activating factor 1 (Apaf-1), promotes activation of caspase-9 (Li et al., 1997; Srinivasula et al., 1998), which then activates downstream caspases, such as caspase-3 and -7 (Budihardjo et al., 1999). Although loss of mitochondrial integrity can also be induced by death receptors, it is not essential for their induction of apoptosis (Strasser et al., 1995).
Indispensable for the regulation of mitochondrial integrity are proteins of the Bcl-2 family. These consist of anti-apoptotic members, such as Bcl-2, Mcl-1, and Bcl-XL and pro-apoptotic members, such as Bad, Bim, and Bid (for review see Adams and Cory, 1998). One mechanism by which cytokines are believed to promote survival is by inhibiting transcription (Dijkers et al., 2000a; Shinjyo et al., 2001) or activity (del Peso et al., 1997; Songyang et al., 1997) of pro-apoptotic members, as well as transcriptionally upregulating anti-apoptotic members (Chao et al., 1998; Kuribara et al., 1999).
Protein kinase B (PKB), also known as c-akt, is regulated by agonist-induced phosphatidylinositol 3-kinase (PI3K) activation, and has been proposed to regulate cytokine-mediated cell survival (Ahmed et al., 1997; Songyang et al., 1997; Eves et al., 1998). Anti-apoptotic signals from PKB include upregulation of Mcl-1 (Wang et al., 1999) and inhibitory phosphorylation of Bad (Songyang et al., 1997), although the relevance of Bad phosphorylation for the survival of hematopoietic cells remains unclear (Scheid and Duronio, 1998). A recently identified mechanism by which PKB can promote rescue from apoptosis is through inhibitory phosphorylation of the forkhead transcription factor FKHR-L1 (FOXO3a) (Brunet et al., 1999; Dijkers et al., 2000a). Activity of this transcription factor has been linked to the induction of apoptosis in hematopoietic cells (Brunet et al., 1999; Dijkers et al., 2000a, b). Although it has been demonstrated in several systems that PKB can mediate rescue from apoptosis, it is not clear whether PKB exerts its anti-apoptotic effect upstream (Kennedy et al., 1999) or downstream (Zhou et al., 2000) of mitochondria. Furthermore, little is known concerning the mechanisms by which activation of FKHR-L1 can lead to induction of the apoptotic program.
Here, we investigate the mechanisms of cytokine withdrawal and forkhead-induced apoptosis and the role of PKB in rescue from apoptosis in cytokine-deprived cells. Our data demonstrate that FKHR-L1, as well as cytokine withdrawal, induce apoptosis through a death receptorindependent pathway. This involves transcriptional upregulation of the pro-apoptotic Bcl-2 family member Bim, loss of mitochondrial integrity, cytochrome c release, and caspase activation. Thus, PKB can protect cells from cytokine withdrawalinduced apoptosis by inhibiting FKHR-L1, resulting in the maintenance of mitochondrial integrity. These data shed new light on the mechanisms by which cytokines, through regulation of PI3K activity, can modulate the survival of hematopoietic lineages.
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Results |
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Cells were either cytokine-starved or treated with 4-OHT for 24 h and apoptosis was measured by analyzing binding of annexin V-FITC. Cells that are annexin V positive represent early apoptotic cells, whereas cells that are stained for both annexin V and propidium iodide (PI) represent cells that have initiated the apoptotic program for a longer period of time. Cells that are dead through necrosis are PI positive and annexin V negative. Both cytokine withdrawal and FKHR-L1 activity induced apoptosis to a similar degree (Fig. 1 A, top and middle; IL-3 withdrawal: 25% ± 4%; 4-OHT addition: 33% ± 4%). Next, we analyzed the kinetics by which apoptosis was induced using DNA laddering, a measure for the final events characterizing apoptosis. Both cytokine withdrawal and FKHR-L1 activity induced apoptosis within a similar time frame (Fig. 1 A, bottom). Recently, we have demonstrated that both p27KIP1 and Bim are transcriptional targets of FKHR-L1 (Dijkers et al., 2000a,b). We examined whether the kinetics of upregulation of p27KIP1 and Bim protein correlated with induction of apoptosis. Both cytokine withdrawal and FKHR-L1 activation resulted in an upregulation of p27KIP1 and Bim (Fig. 1, B and C). These events occurred relatively early and preceded the cleavage of DNA observed in Fig. 1 A.
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Cytokine withdrawal and FKHR-L1 activity induce cell cycle arrest followed by apoptosis
p27KIP1 is involved in cell cycle arrest in G1 through the inhibition of cyclinCDK complexes (Polyak et al., 1994; Toyoshima and Hunter, 1994), but has also been described to function in the induction of apoptosis through a yet unidentified mechanism (Wang et al., 1997; Dijkers et al., 2000a). To see whether upregulation of p27KIP1 reflected an altered distribution of cells in the cell cycle, we analyzed the cell cycle profile of cells at various time points. Upon cytokine withdrawal, cells stopped initiating cell division and accumulated in G1, within the first 8 h of starvation (Fig. 3 A). After 16 h of cytokine deprivation cells started to undergo apoptosis, as measured by cells having a DNA content <2n chromosomes, the sub-G1 peak. By 48 h, a majority of cells had initiated a program of apoptotis (58% ± 5%). Similar findings were observed in 4-OHTtreated FKHR-L1(A3):ER* cells (Fig. 3 B; 48 h, 49% ± 3%), suggesting that the presence of a G1 arrest before the onset apoptosis is probably related to the initial upregulation of p27KIP1 (Fig. 1, B and C).
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To determine whether FKHRL1-DBD could indeed inhibit FKHR-L1mediated transcription, COS cells were transiently transfected with the pGL2-p27kip luciferase promoter construct together with a constitutively active FKHR-L1(A3) mutant and increasing concentrations of the inhibitory mutant FKHRL1-DBD. As shown in Fig. 8 A, FKHR-L1(A3) strongly induced pGL2-p27kip promoter activity, which was inhibited by FKHRL1-DBD in a concentration-dependent manner. Similar results were shown for the pGL26xDBE construct, which contains six consecutive FKHR-L1 binding sites. Again, FKHRL1(A3) strongly induced promoter activity, which was inhibited by increasing concentrations of FKHRL1-DBD.
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To determine then whether inhibition of forkhead-related transcription factors (FKHR-L1, FKHR, and AFX) could protect cells from cytokine withdrawalinduced apoptosis, Ba/F3 cells were transfected with spectringreen fluorescence protein (GFP) plus a control vector, FKHRL1-DBD, or constitutively active PKB (gagPKB). Cells were cultured without IL-3 and levels of apoptosis were measured by annexin V binding, as described in the Materials and methods. Cells transfected with the control vector exhibited the same level of apoptosis as untransfected cells (Fig. 8 C, top). However, cotransfection of FKHRL1-DBD (Fig. 8 C, middle) dramatically reduced the level of apoptosis compared with untransfected cells (17% vs. 36%, respectively). This rescue from apoptosis was comparable with that observed in cells transfected with gag-PKB (Fig. 8 C, bottom). Taken together with our previous findings (Dijkers et al., 2000a,b), these data demonstrate that FKHR-L1 is indeed an important component in regulating the initiation of the apoptotic program.
Inhibition of Bim expression is important for PI3K-mediated rescue from apoptosis
To examine the relevance of inhibition of Bim expression for PI3K/PKBmediated rescue from apoptosis in vivo, we used hematopoietic stem cells isolated from either wild-type mice or mice lacking both Bim alleles (Bim-/-; Bouillet et al., 1999). Bone marrow Sca1+ stem cells were cultured either with cytokines, without cytokines, or with cytokines as well as the PI3K inhibitor LY294002. Cells were subsequently analyzed after 24 h by annexin V staining, or after 48 h by Rh-123 binding (Fig. 9). In cells cultured with cytokines, the level of apoptosis, as measured by annexin V or Rh-123, is low and exhibits little difference between mice. Upon removal of survival factors, cells isolated from the Bim-/- mice have a significantly enhanced survival advantage, demonstrating that Bim is indeed critical in the regulation of hematopoietic stem cell survival. This is particularly apparent as measured by Rh-123 after 48 h. To determine if inhibition of Bim levels is a critical component of the mechanism by which PI3K/PKB regulates cell survival, stem cells were treated with LY294002. Treatment of cells isolated from wild-type mice with this PI3K inhibitor induced apoptosis to a level at least equal to that observed upon cytokine withdrawal (Fig. 9). However, in Bim-/- mice, the percentage of apoptotic cells observed after LY294002 treatment was significantly reduced compared with wild-type, particularly when mitochondrial integrity was analyzed after 48 h (Fig. 9 B). These data suggest that Bim is a critical downstream target of PI3K action.
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Discussion |
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We also analyzed whether Fas/FasL signaling may be involved in the induction of apoptosis upon cytokine withdrawal as previously proposed (Brunet et al., 1999). Neither cytokine withdrawal nor FKHR-L1 activity resulted in caspase-8 cleavage, an event specific for death receptor signaling (Juo et al., 1998; Varfolomeev et al., 1998). In addition, cytokine withdrawal had no effect on FasL promoter activity (Fig. 2 D) or FasL protein expression (unpublished data). Importantly, treatment of Ba/F3 cells with FasL did not induce apoptosis, suggesting a lack of a functional Fas/FasL death receptor signaling pathway. This suggests that apoptosis induced either by cytokine withdrawal, PI3K inhibition, or FKHR-L1 activity is initiated through a death receptorindependent mechanism. In support of this, overexpression of anti-apoptotic Bcl-2 members, which rescue death receptorindependent apoptosis, but not death receptordependent apoptosis in lymphocytes (Itoh et al., 1993; Scaffidi et al., 1998), are able to rescue both cytokine withdrawal as well as FKHR-L1induced apoptosis (Chao et al., 1998; Dijkers et al., 2000b). Interestingly, although the FasL promoter was not activated by cytokine withdrawal, a small region of this promoter previously shown to contain three forkhead binding sites was (Brunet et al., 1999; Fig. 2 D). This suggests that in the context of the intact FasL promoter, secondary factors are responsible for mediating promoter activity. Possibly, there are critical forkhead cofactors absent in Ba/F3 cells, or other factors are expressed that actively repress FasL promoter activity. This might explain the differences between our data and that previously described by Brunet et al. (1999) in T cells.
PKB has been demonstrated to negatively regulate members of a subfamily of forkhead transcription factors: AFX, FKHR, and FKHR-L1 (for review see Datta et al., 1999). Recently, members of the SGK (serum and glucocorticoid-induced kinases) family, which phosphorylate consensus sequences similar to PKB, were found to be required for full phosphorylation of FKHR-L1 in vivo and in IL-3mediated survival (Liu et al., 2000; Brunet et al., 2001). This suggests that both kinases may be required for phosphorylation-mediated inactivation of FKHR-L1, and may explain why PKB was unable to completely inhibit cytokine withdrawalinduced apoptosis (Fig. 7). However, PKB was capable of significantly abrogating cytokine withdrawalinduced loss of mitochondrial potential (Fig. 7 C). Thus, we can conclude that PKB exerts its anti-apoptotic effect at a premitochondrial level, preventing intracellular release of cytochrome c. A potential role for PKB in rescue from apoptosis and prevention of cytochrome c leakage has also been proposed in apoptosis induced in Rat1 fibroblasts by UV irradiation (Kennedy et al., 1999), as well as in apoptosis induced in epithelial cells by detachment from the extracellular matrix (Rytomaa et al., 2000). However, PKB has also been previously shown to inhibit ceramide-induced apoptosis in hybrid neuron motor 1 cells downstream of cytochrome c release (Zhou et al., 2000). These findings may be explained by differences in apoptotic stimuli in different cell types, and indicates that PKB has the potential to act at multiple levels. Furthermore, difference in species could be an explanation for the differential contribution of PKB in rescue from apoptosis. PKB has been suggested to promote rescue from apoptosis by inhibitory phosphorylation of caspase-9 in human cells (Cardone et al., 1998), but not in mouse or rat cells because the PKB phosphorylation site in caspase-9 is not present (Fujita et al., 1999). PKB has also been linked to the upregulation of the anti-apoptotic Bcl-2 member Mcl-1 (Wang et al., 1999), which is essential in cytokine-mediated rescue from apoptosis (Chao et al., 1998). This regulation of an anti-apoptotic Bcl-2 member, involved in the maintenance of mitochondrial integrity, also supports a role for PKB upstream of cytochrome c leakage in cytokine-mediated rescue from apoptosis.
The p21ras-activated protein kinase MEK has also been proposed to rescue cells from apoptosis (Perkins et al., 1996; Shimamura et al., 2000), potentially through activation of downstream targets that phosphorylate Bad (Shimamura et al., 2000). Furthermore, MEK-initiated signals can result in the phosphorylation of anti-apoptotic members of the Bcl-2 family, thereby enhancing their stability (Breitschopf et al., 2000). However, using the myrPKB:ER* cell line, we have demonstrated that PKB alone is sufficient to protect cells from apoptosis (Fig. 6, B and C). Our data do not, however, rule out the possibility that MEK plays a role in these events.
Increased PKB activity can result in cellular transformation (Bellacosa et al., 1991; Cheng et al., 1992; Haas-Kogan et al., 1998), although the exact mechanisms by which PKB is capable of promoting oncogenesis remains to be established. Inhibitory phosphorylation of FKHR-L1 could very well contribute to this process, leading to a decrease in both Bim and p27KIP1 levels. This is supported by the observation that a decrease in p27KIP1 levels is associated with a poor prognosis in cancer (Catzavelos et al., 1997; Loda et al., 1997; Ohtani et al., 1999).
Two critical experiments have demonstrated that FKHR-L1 is a critical effector of cell death induced by cytokine withdrawal, and that Bim is an important downstream target of PI3K/PKB action. First, in Fig. 8 we demonstrate that ectopic expression of an inhibitory FKHR-L1 significantly reduces the levels of apoptosis observed after cytokine withdrawal. This is similar to the effect of expressing a constitutively active mutant of PKB. Although inhibition of apoptosis is dramatic, it is not complete. This could either be due to additional pro-apoptotic pathways, or simply that the levels of expression of these transfected proteins is relatively low in Ba/F3 cells. Using Bim knockout mice, we demonstrate that hematopoietic stem cells isolated from these animals have much reduced levels of apoptosis compared with wild-type mice upon cytokine withdrawal. This is in agreement with observations previously made in leukocytes isolated from these mice (Bouillet et al., 1999). If inhibition of Bim transcriptional levels is indeed critical for PI3K/PKBmediated cell survival, it follows that inhibition of PI3K activity would have a significantly reduced effect on apoptosis in cells isolated from Bim-deficient mice. Indeed our data demonstrate this to be the case (Fig. 9). Thus, it appears that in cytokine-dependent cells, repression of Bim expression may be one of the major mechanisms by which PI3K/PKB signaling results in enhanced cell survival in vivo. It should, however, be noted that the cytoprotective effects of FKHRL1-DBD and Bim knockout are not complete. This suggests the possibility that there are other targets and mechanisms contributing to the induction of apoptosis upon cytokine withdrawal. Similarly, it has not been shown that FKHR-L1 is critical for the induction of Bim upon cytokine withdrawal.
Taken together, our data suggest that cytokine-induced signaling can inhibit cells from apoptosis through activation of PKB (or possibly SGK), which inhibits FKHR-L1 and Bad through phosphorylation and transcriptionally upregulates Mcl-1. In the absence of cytokines, PKB is inactive, resulting in dephosphorylation and activation of Bad and transcription of FKHR-L1 targets p27KIP1 and Bim. This results in induction of the apoptotic program through loss of mitochondrial integrity, leakage of cytochrome c, subsequent activation of caspases, and cleavage of substrates. The events mediating cytokine withdrawalinduced apoptosis, as well as, cytokine-mediated rescue from apoptosis are summarized in Fig. 10.
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Materials and methods |
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Mice deficient for Bim have been described previously (Bouillet et al., 1999). Each Bim-/- mouse used in this study was produced by crossing (C57bl.6J background) and genotyped by PCR analysis. All Bim-/- mice were compared with age-matched wild-type littermates (34 mo old). After sacrifice, the femurs were removed and bone marrow was flushed out with Iscove's modified DuIbecco's medium (IMDM) containing 20% (vol/vol) FCS, penicillin, and streptomycin through a MACS prefilter (Miltenyi Biotec). An aliquot was removed, and nucleated cells were counted with an improved Neubauer chamber after lysing the red cells in 1% (vol/vol) acetic acid. The remaining cells were pelleted and incubated for 15 min at 4°C with 200 ml Sca1 MultiSort microbeads per 108 cells. Sca1+ cells were isolated through two columns, either manually (MACS-MS columns) or with an AutoMACS machine, according to the manufacturer's protocol. The Sca1+ cells were resuspended in Iscove's modified Dulbecco's medium (IMDM) containing 20% (vol/vol) FCS, penicillin, and streptomycin at 106 cells/ml, and their purity was determined as around 95% by flow cytometry. The Sca1+ cells were isolated for 67 d in the presence of IL-3 (10 ng/ml), IL-6 (10 ng/ml), and stem cell factor (100 ng/ml), and fresh medium containing cytokines was added as necessary (Kurata et al., 1998).
Antibodies and reagents
Polyclonal antibodies against cleaved caspase-3 (#9661), cleaved caspase-7 (#9491S), and PARP (#9542) and phospho-Ser473 PKB (#9271S) were from New England Biolabs, Inc. Polyclonal caspase-8 antibody (559932) and monoclonal cytochrome c antibody (clone 7H8.2C12) were from BD PharMingen. Bim polyclonal antibody was purchased from Affinity BioReagents, Inc. p27KIP1 and RACK1 mAb were purchased from Transduction Laboratories. PI was from Sigma-Aldrich. The annexin V-FITC kit and SUPERFas Ligand were from Alexis Biochemicals Corp. Rh-123 was purchased from Molecular Probes. All other chemicals were reagent grade. pSG5mycFKHRL1(DBD) was made by PCR cloning nucleotides corresponding to amino acids 141268 from FKHR-L1(A3) in-frame into pSG5myc. FasL promoter and 6xDBE luciferase reporter constructs were provided by Anne Brunet (Children's Hospital, Boston, MA) and Boudewijn (University Medical Center, Utrecht, Netherlands), respectively.
DNA laddering
107 cells were treated as indicated, lysed on ice for 10 min in buffer A (10 mM Tris-Cl, pH 7.4, 10 mM EDTA, 0.2% TX-100, supplemented with 1 mM PMSF, 0.1 mM aprotinin, and 1 mM leupeptin), and centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was added to an equal volume phenol/chloroform, rocked gently for 10 min, centrifuged, and the upper phase was added to 1/10 vol sodium acetate (3 M, pH 5.4) and 2.5 vol ethanol, incubated at -20°C for 15 min, and subsequently spun down. The pellet was air dried, resuspended in TE containing 2 µg/ml RNase A, incubated at 37°C for 30 min, and run on a 2% agarose gel.
Western blotting
For the detection of all proteins, cells were lysed in ELB buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM EDTA together with inhibitors) (Medema et al., 1998). Protein concentration was measured and equal amounts of protein were analyzed by SDS-PAGE. Blots were incubated overnight at 4°C with appropriate antibodies (1:1,000), and after hybridization with secondary antibodies, they were developed using ECL (Amersham Pharmacia Biotech).
FACS® analysis of apoptosis
Preparation of cells for the analysis of cell cycle profiles has been described previously (Dijkers et al., 2000a). For the analysis of apoptosis by annexin V staining, cells were washed with ice cold PBS and resuspended in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Cells were then incubated with FITC-conjugated annexin V (Bender Medsystems) for 10 min at room temperature, washed, and resuspended in binding buffer containing 1 µg of PI/ml. Fluorescence was analyzed by FACS®.
For transient transfections, Ba/F3 cells were electroporated (0.28 kV; capacitance, 960 µF), and 2 h after electroporation, dead cells were removed by separation through a Ficoll gradient (2,500 rpm for 20 min). Cells were harvested 48 h after electroporation and analyzed by FACS® as described above, but using annexin Vphycoerythrin.
Analysis of mitochondrial depolarization
For the analysis of changes in mitochondrial potential, m (Ferlini et al., 1996), cells were incubated in RPMI together with 10 µg/ml Rh-123 (Molecular Probes) at 37°C for 30 min, washed twice with PBS, and analyzed by FACS® (Dijkers et al., 2000b). The percentage of cells falling within the range of Rh-123 fluorescence, indicative of depolarized cells, is shown.
Analysis of intracellular cytochrome c release from mitochondria
Mitochondria and cytosol fractions were obtained using the mitochondria/cytosol fractionation kit (Kordia) according to the manufacturer's protocol. In brief, 5 x 107 cells were treated as indicated, washed once in PBS, resuspended in 500 µl cytosol extraction buffer, and incubated on ice for 10 min. Subsequently, cells were homogenized in an ice cold tissue grinder. The homogenate was centrifuged at 700 g for 10 min at 4°C. The remaining supernatant was then centrifuged at 10,000 g for 30 min at 4°C. Subsequently, the supernatant was collected as the cytosolic fraction. The pellet was resuspended in 100 µl mitochondrial extraction buffer and vortexed for 10 s (mitochondrial fraction). Protein concentration was measured and equal amounts of protein were analyzed by SDS-PAGE. The amount of cytochrome c in the different fractions was determined by Western blotting using a monoclonal antibody against cytochrome c.
Luciferase assays
For transient transfections, Ba/F3 cells were electroporated (0.28 kV; capacitance, 960 µF) with 20 µg of a luciferase reporter plasmid containing the FasL enhancer region (Holtz-Heppelmann et al., 1998) or a luciferase reporter containing the portion of the FasL promoter that contains three forkhead binding sites (FHRE-LUC reporter) (Brunet et al., 1999). Cells were cotransfected with 50 ng of a renilla luciferase plasmid (pRL-TK; Promega) to normalize for transfection efficiency. After transfection, cells were cultured with or without IL-3 for 18 h. Cells were then harvested, lysed in commercially available luciferase lysis buffer, and luciferase activity was determined. In some experiments, a luciferase reporter containing the p27KIP1 promoter was used.
COS cells were transiently transfected with the pGL2-p27kip luciferase promoter construct or a pGL26xDBE luciferase construct (containing six FKHR-L1 binding sequence), together with pECE-HA-FKHR-L1(A3), pSG5-myc-FKHRL1-DBD expression, or control vectors and the internal transfection control (pRL-TK) by calcium phosphate precipitation. Values were corrected for transfection efficiency and represent the mean of at least three independent experiments (± SEM).
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Footnotes |
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* Abbreviations used in this paper: 4-OHT, 4-hydroxy tamoxifen; DBD, DNA-binding domain; FasL, Fas ligand; GFP, green fluorescence protein; IL, interleukin; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; Rh-123, rhodamine-123; SGK, serum and glucocorticoid-induced kinases.
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Acknowledgments |
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Kim U. Birkencamp is supported by the Netherlands Organization for Scientific Research. Eric W.-F. Lam is supported by the Cancer Research Campaign and Leukemia Research Foundation. N. Shaun B. Thomas is supported by the Leukemia Research Foundation and the Sir Charles Wolfson Trust.
Submitted: 17 August 2001
Revised: 6 December 2001
Accepted: 11 December 2001
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