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¹ Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
² Cancer Chemotherapy Center, Japanese foundation for Cancer Research, Koto-ku, Tokyo 135-8550, Japan
³ Okazaki Institute for Integrative Biosciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan
⁴ The Burnham Institute for Biomedical Research, 10901 N. Torrey Pines Rd, La Jolla, CA 92037, USA

	Abstract

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Cellular FLIP (cFLIP) is a homologue of caspase-8 without protease activity that inhibits the apoptosis signaling initiated by death receptor ligation. We previously reported that a long form of cFLIP (cFLIP-L) inhibits ubiquitylation of ß-catenin and enhances Wnt signaling. Here we show that cFLIP-L impairs the function of the ubiquitin-proteasome system (UPS), and increases the accumulation of various short-lived proteins, such as GFP conjugated with destabilization sequence, ß-catenin and HIF1, that are subjected to rapid ubiquitylation and degradation by proteasomes. Accordingly, ß-catenin- and HIF1-mediated gene expressions are induced in the cFLIP-L-expressing cells. Exogenously expressed cFLIP-L accumulates in aggregates at the peri-nuclear region in the cells, and the cFLIP-L aggregates are refractory to solubilization. Like exogenously expressed cFLIP-L, the endogenous cFLIP in A549 lung cancer cells displays particulate distribution in the cells and more than 60% of cFLIP-L is refractory to solubilization. Down-regulation of cFLIP in A549 cells by RNA-mediated interference reduced ß-catenin- and HIF1-mediated gene expression. These results suggest that cFLIP-L is prone to aggregate and impairs UPS function, which could be involved in the pathological function of cFLIP-L expressed in certain cancer cells.

	Introduction

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The highly conserved ubiquitin-proteasome system (UPS) controls the stability of many cellular proteins (Rock et al. 1994; Lee & Peter 2003). These include ß-catenin and HIF1, the expression of which in cytoplasm is normally maintained at a low level by rapid degradation mediated by the UPS (Ikeda et al. 1998; Kitagawa et al. 1999; Tanimoto et al. 2000). When cells are stimulated by Wnt ligand or hypoxia, the ubiquitylation of ß-catenin or HIF1, respectively, is inhibited, resulting in accumulation of the proteins (Forsythe et al. 1996; Polakis 2000). The accumulated ß-catenin and HIF1 translocate to the nucleus and induce expression of target genes to promote cell proliferation and angiogenesis, respectively. Short-lived proteins containing a RING finger domain, such as XIAP and Siah1, are also subjected for UPS-mediated degradation (Matsuzawa & Reed 2001; Suzuki et al. 2001). The RING domain recruits a ubiquitin-conjugating enzyme (E2), and mediates the ubiquitylation of XIAP and Siah1 (Lorick et al. 1999).

In the UPS, ubiquitin is covalently attached to a lysine residue of a substrate protein by a sequential reaction mediated by an activating enzyme (E1), a conjugating enzyme (E2) and protein ligases (E3) (Scheffner et al. 1995). The addition of four or more ubiquitin moieties targets the substrates protein for destruction via the proteasome. Abnormality of the UPS leads to various diseases, such as neurodegenerative disorders, hereditary diseases and cancer (Lee & Peter 2003). Several groups have reported that protein aggregates accumulated in inclusion bodies profoundly impair the functional capacity of the UPS (Kopito 2000; Bence et al. 2001). In an in vitro model, however, the UPS was impaired in the absence of large inclusion bodies, suggesting that intermediate forms or smaller protein aggregates may be able to inhibit UPS function (Bennett et al. 2005).

Cellular FLIP (c-FLIP, also known as I-FLICE, FLAME-I, Casper, CASH, MRIT and Usurpin) is an inhibitor of apoptosis initiated by death receptor ligation (Irmler et al. 1997). The long form of c-FLIP (cFLIP-L) is highly homologous to caspase-8, containing two death effector domains (DED) and a caspase-like domain at the amino and carboxy terminal, respectively. cFLIP-L, however, does not have a caspase activity due to the lack of a conserved cysteine residue in the caspase-like domain. Upon death receptor ligation, cFLIP-L is recruited to the death receptor complex, together with FADD and caspase-8, and inhibits apoptosis signaling. In addition to the apoptosis inhibition, cFLIP-L mediates the activation of NF-B, PI3 K and Erk by virtue of its capacity to recruit the adaptor proteins involved in each signaling pathway, such as TRAF-1, TRAF-2, RIP and Raf-1 (Chaudhary et al. 2000; Kataoka et al. 2000; Fang et al. 2004; Kataoka & Tschopp 2004). The inhibition of JNK pathway by direct binding to MKK7 was also reported (Nakajima et al. 2006). Homozygous disruption of c-FLIP gene in mice results in a failure of heart development at embryonic day 11.5, suggesting an essential role of cFLIP in cardiac development during embryogenesis (Yeh et al. 2000).

We and others previously reported that cFLIP-L inhibits ubiquitylation of ß-catenin, and enhances Wnt signaling (Naito et al. 2004; Nakagiri et al. 2005). In an effort to clarify the mechanism how cFLIP-L inhibits the ubiquitylation of ß-catenin, we found that cFLIP-L is prone to aggregate and impairs UPS function, thereby increasing the accumulation of various short-lived proteins, including ß-catenin.

	Results

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Elevation of various protein levels and their signaling by cFLIP-L

cFLIP-L increases endogenous cytosolic ß-catenin and enhances Wnt signaling (Naito et al. 2004). To examine the specificity of a cFLIP-L-mediated elevation of protein levels, expression plasmids encoding various proteins were co-transfected with cFLIP-L (Fig. 1). Co-expression of cFLIP-L increased HIF1, ß-catenin, XIAP and Siah-1, that are known to be degraded by the UPS. Consistent with the elevation of the protein level, cFLIP-L increased gene expression mediated by co-transfected HIF1 (Fig. 2A) and ß-catenin (Fig. 2B). cFLIP-L also induced gene expression mediated by endogenous HIF1 (Fig. 2A) and ß-catenin (Fig. 2B), suggesting the accumulation of the endogenous proteins in the cFLIP-L-expressing cells. These results indicate that cFLIP-L increases the level of various short-lived proteins degraded by the UPS.

View larger version (20K): [in this window] [in a new window]   Figure 1  cFLIP-L increases various short-lived proteins. Expression plasmids encoding Myc-tagged cFLIP-L and either V5-tagged HIF-1 (A), HA-tagged ß-catenin (B), FLAG-tagged XIAP (C) or HA-tagged Siah-1 (D) were transfected into cells. Some cells were treated with MG-132 (20 µM) for 5 h before being harvested. Whole-cell lysates (A, C, D) or cytosolic fractions (B) were analyzed by Western blot with the indicated antibodies.

View larger version (7K): [in this window] [in a new window]   Figure 2  Activation of gene expressions by cFLIP-L. Cells were transfected with plasmids encoding Myc-tagged cFLIP-L, pRL-TK together with the reporter plasmids 5xHRE/pGL3/VEGF/E1b (A), TOP-TK-Luc (B: upper panel) or FOP-TK-Luc (B: lower panel), as described in Experimental procedures. The luciferase activity was measured with the Dual-Luciferase Reporter Assay System and expressed as the fold increase compared with the level observed in control cells without cFLIP-L. The data represent the means of triplicate determinations. The error bars indicate standard deviations. *P < 0.05, **P  < 0.001 by Welch's t-test.

We next examined the effect of cFLIP-L on the ubiquitylation of the proteins. Cells were transfected with plasmids encoding HA-tagged ubiquitin and Flag-tagged XIAP, and cell lysates were prepared. The XIAP was immunoprecipitated with anti-Flag antibody, and the resulting immune complexes were analyzed by immunoblotting with anti-HA antibody to detect ubiquitylated XIAP (Fig. 3A), which migrated as a smear of protein bands with slower mobility in gels. The polyubiquitylated XIAP was increased by the treatment with the proteasome inhibitor MG-132 (Fig. 3A, lane 4), and was dramatically suppressed by the co-expression of cFLIP-L (lane 5). Similar results were obtained when Flag-tagged ß-catenin was used (Fig. 3B). These results indicate that cFLIP-L inhibits ubiquitylation of these proteins.

View larger version (20K): [in this window] [in a new window]   Figure 3  cFLIP-L inhibits ubiquitylation. Cells were transfected with expression plasmids encoding Myc-tagged cFLIP-L, HA-tagged ubiquitin together with Flag-tagged XIAP (A) or FLAG-tagged ß-catenin (B), as indicated. Some cells were treated with MG-132 (20 µM) for 5 h before being harvested. The cell lysates were immunoprecipitated and the precipitates were analyzed by Western blot with the indicated antibodies. Aliquots of the cell lysates were examined for the expression of Myc-tagged cFLIP-L. The lower molecular weight protein band in lane 5 of the middle panel in (A) is a fragment of XIAP that is cleaved by caspase-3 activated by cFLIP-L expression.

To study the mechanism by which cFLIP-L inhibits the ubiquitylation of various proteins, we examined the subcellular localization of cFLIP-L and ubiquitin/ubiquitylated proteins. Immunostaining of the cFLIP-L showed that cFLIP-L accumulated in aggregates at the peri-nuclear region in the cells, and the signal with multi-ubiquitin immunostaining (Fujimuro et al. 1994; Lelouard et al. 2002) co-localized with the aggregated cFLIP-L (Fig. 4A). The co-localization of ubiquitin with cFLIP-L was also observed in the cells expressing GFP-ubiquitin and cFLIP-L (Fig. 4B). cFLIP-L was robustly ubiquitylated (Fig. 4C, lane 2), but the ubiquitylation was not enhanced by MG-132 treatment (lane 3), suggesting that the ubiquitylated cFLIP-L is not degraded efficiently by proteasomes, probably due to the aggregate formation. Biochemical fractionation of the cFLIP-L-expressing cells into cytoplasmic, nuclear and insoluble fractions showed that transiently expressed cFLIP-L was mostly in the insoluble fraction (Fig. 4D, lane 4). These results suggest that the cFLIP-L which is prone to aggregate in the cells is refractory to solubilization.

View larger version (31K): [in this window] [in a new window]   Figure 4  cFLIP-L is ubiquitylated and forms aggregates refractory to solubilization. HT-1080 cells (A) or HT-1080 cells constitutively expressing GFP-tagged ubiquitin (B) were transfected with expression plasmids encoding Myc-tagged cFLIP-L in the presence of 20 µM Z-VAD-fmk. After 24 h, the cells were fixed and immunostained with the indicated antibodies. Arrows indicate the cells containing aggregated cFLIP-L. Bars: 50 µm. (C) Cells were transfected with HA-tagged ubiquitin and Myc-tagged cFLIP-L in the presence of 20 µM Z-VAD-fmk for 24 h. The whole cell lysates were immunoprecipitated with anti-Myc and the precipitates were analyzed by Western blot with indicated antibodies. Aliquots of the cell lysates were Western blotted with the indicated antibodies. (D) HT-1080 cells transiently transfected with Myc-tagged cFLIP-L were fractionated with NE-PER Nuclear and Cytoplasmic Extraction Reagents, and the fractions of same cell number were analyzed by Western blot with indicated antibodies. C, cytosol fraction; N, nuclear fraction; P, precipitated insoluble fraction. Whole cell lysates (W) were loaded on the same gel as a control.

Since protein aggregates impair UPS function (Bence et al. 2001; Bennett et al. 2005), we next examined whether the cFLIP-L aggregate impairs UPS function by using a GFP-degron reporter protein (Fig. 5). GFP-degron contains a highly ubiquitylation-prone sequence (CL1-degron) at the carboxy-terminal (Gilon et al. 1998), and therefore, is targeted for efficient clearance by the UPS (Bence et al. 2001; Bennett et al. 2005). The expression of GFP-degron was lower than that of GFP, which was increased to the level of GFP by the proteasome inhibitor MG132, suggesting GFP-degron is targeted for degradation by the UPS in cells. Co-expression of cFLIP-L increased the expression of GFP-degron as well as MG132 (Fig. 5A). To further examine the impairment of the UPS by cFLIP-L, cells constitutively expressing GFP-degron were treated with a protein-synthesis inhibitor, and the GFP-degron level was measured by Western blot analysis. cFLIP-L inhibited the decline of GFP-degron as did MG132 (Fig. 5B,C). The increment of GFP-degron was observed by the expression of a lower amount of cFLIP-L that was comparable to the endogenous cFLIP-L expressed in A549 lung cancer cells and ACHN renal carcinoma cells (Fig. 5D). These results indicate that cFLIP-L inhibits the UPS.

View larger version (26K): [in this window] [in a new window]   Figure 5  cFLIP-L impairs UPS function. (A) HT-1080 cells were transfected with expression plasmids encoding Myc-tagged cFLIP-L and either GFP or GFP-degron. Some cells were treated with MG-132 (20 µM) for 5 h before harvest. Whole cell lysates were Western blotted with the indicated antibodies. (B) HT-1080 cells constitutively expressing GFP-degron were transfected with Myc-tagged cFLIP-L. After 24 h, the cells were treated with 50 µg/mL of cycloheximide for the indicated times, and the whole cell lysates were analyzed by Western blotting with the indicated antibodies. (C) The GFP-degron bands shown in (B) were measured and expressed as percentages compared with the corresponding protein level at time zero. (D) HT-1080 cells constitutively expressing GFP-degron were transfected with the indicated amount of Myc-tagged cFLIP-L, and whole cell lysates were Western blotted with the indicated antibodies. Whole cell lysates from A549 and ACHN cells were loaded on the same gel to compare the cFLIP-L level.

View larger version (30K): [in this window] [in a new window]   Figure 6  UPS impairment and localization of cFLIP mutants. Myc-tagged cFLIP mutants were expressed in HT-1080 cells, and whole cell lysates were analyzed by Western blot (A) or the transfected cells were immunostained with anti-Myc antibody (B). FLIP-L: wild-type cFLIP-L; FLIP (1–438): cFLIP-L that has deleted C-terminal 42 amino acids; FLIP-DED A/B: cFLIP-L that has deleted caspase-like domain; FLIP-Casp: cFLIP-L that has deleted DEDs; FLIP-L (D376A): D376 that is a caspase-8 site in the caspase-like domain of cFLIP-L was substituted to A; FLIP-L (F25G/F114G): F25 and F114 that are conserved in DEDs were substituted to G. Bar: 20 µm.

To examine if cFLIP-L directly inhibits proteasome activity, we also established a cell line expressing ZsGreen-ODC that directly binds to the proteasome by ornithine-decarboxylase (ODC) sequence, and therefore, is degraded by the proteasome without ubiquitylation. The expression of ZsGreen-ODC was increased by MG132, but not by cFLIP-L (Fig. 7), indicating that cFLIP-L does not directly inhibit proteasome activity. Taken together with the inhibition of ubiquitylation by cFLIP-L (Fig. 3), these results indicate that cFLIP-L impairs the UPS by inhibiting ubiquitylation of proteins, not by directly inhibiting the proteasome.

View larger version (18K): [in this window] [in a new window]   Figure 7  Proteasome activity is not inhibited in cFLIP-L-expressing cells. HT-1080 cells constitutively expressing ZsGreen-ODC were transfected with expression plasmids encoding Myc-tagged cFLIP-L, and the whole cell lysates were analyzed by Western blot with the indicated antibodies.

To study the subcellular localization and function of endogenous cFLIP, we fractionated A549 and ACHN cells into cytoplasmic, nuclear and insoluble fractions. While cFLIP-L in ACHN cells was found mostly in the cytoplasmic fraction (Fig. 8A, right panel), more than 60% of cFLIP-L was recovered in the insoluble fraction in A549 cells (Fig. 8A, left panel), which was reminiscent of the transiently expressed cFLIP-L: more than 90% of cFLIP-L was in the insoluble fraction (Fig. 4D). Immunocytochemical analysis revealed particulate distribution of cFLIP protein in A549 cells and diffused staining in ACHN cells (Fig. 8B). These results indicate that endogenous cFLIP-L in A549 cells accumulates in small aggregates in the cells and is refractory to solubilization.

View larger version (18K): [in this window] [in a new window]   Figure 8  Subcellular distribution of endogenous cFLIP-L. (A) A549 lung cancer (left panels) and ACHN renal carcinoma (right panels) cells were fractionated with NE-PER Nuclear and Cytoplasmic Extraction Reagents, and the fractions of the same cell number were analyzed by Western blot with the indicated antibodies. C, cytosol fraction; N, nuclear fraction; P, precipitated insoluble fraction. Whole cell lysates (W) were loaded on the same gel as a control. (B) A549 and ACHN cells were immunostained with anti-FLIP antibody. Bar: 50 µm.

View larger version (18K): [in this window] [in a new window]   Figure 9  Down-regulation of cFLIP in A549 cells reduces gene expression mediated by HIF1 and ß-catenin. (A) Whole-cell lysates from A549 cells that had been transfected with control shRNA vector or c-FLIP shRNA vector were analyzed by immunoblotting with the indicated antibodies. (B) Reduced HIF1 signaling in c-FLIP-depleted cells. A549 cells were transfected with control shRNA vector or FLIP shRNA vector together with 5xHRE reporter gene and pRL-TK as described in Experimental procedures. After 36 h, the cells were treated with 10 mM CoCl2 for 12 h. The luciferase activity was measured with the Dual-Luciferase Reporter Assay System and expressed as the fold increase compared with the level observed in control cells treated without CoCl2. The data represent the means of triplicate determinations. The error bars indicate standard deviations. (C) Reduced Wnt signaling in c-FLIP-depleted cells. A549 cells were transfected with control shRNA vector or FLIP shRNA vector, together with TOP-TK-Luc (left), FOP-TK-Luc (right) and pRL-TK. After 36 h, the cells were treated with conditioned medium containing 500 ng/mL Wnt3a for 12 h. The luciferase activity was measured with Dual-Luciferase Reporter Assay System and expressed as the fold increase compared with the level observed in control cells treated without Wnt3a. The data represent the means of triplicate determinations. The error bars indicate standard deviations. *P < 0.001 by Welch's t-test.

	Discussion

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The DEDs of cFLIP-L play an important role in aggregate formation and UPS impairment, since substitution of the conserved Phe to Gly in DEDs (F25G/F114G) abrogated both activities (Fig. 6). Homotypic DED–DED interaction is reported in most DED-containing proteins including cFLIP-L (Irmler et al. 1997; Tibbetts et al. 2003). When over-expressed, some DEDs oligomerize into filament-like structure called death effector filaments (Siegel et al. 1998; Tibbetts et al. 2003; Park et al. 2005), as shown by the expression of FLIP-DED A/B (Fig. 6B). In cFLIP-L expressing cells, however, we did not observe such filamentous structures but did find aggregates of cFLIP-L protein. Since FLIP-Casp diffuses to the cytoplasm, both DEDs and the Caspase-like domain are required to form aggregates and to impair UPS. The cleavage of cFLIP-L by caspase-8 does not affect the aggregate formation and UPS impairment by cFLIP-L, because a caspase-noncleavable mutant (cFLIP-L (D376A)) exhibited similar activity to wild-type cFLIP-L.

In A549 lung cancer cells, more than 60% of endogenous cFLIP-L was refractory to solubilization, and particulate distribution of cFLIP was observed. Down-regulation of cFLIP in the A549 cells reduced HIF1- and ß-catenin-mediated gene expression, suggesting that the endogenous cFLIP in the A549 cells impairs UPS function, although cFLIP-L is not sequestered into inclusion bodies in the A549 cells. This is in line with the recent report that the impairment of UPS by aggregation-prone proteins precedes inclusion body formation (Bennett et al. 2005). In contrast to the A549 cells, ACHN renal carcinoma cells showed diffused distribution of cFLIP-L, and the protein was efficiently solubilized by detergents. At present, we do not know why cFLIP-L diffuses in the ACHN cells. A binding protein to cFLIP-L may exist in the ACHN cells that affect cFLIP-L distribution, although we do not have any evidences. Silencing of cFLIP-L could not be successfully attained in the ACHN cells, probably due to poor transfection efficiency.

Since cFLIP-L is expressed in various human cancers (Irmler et al. 1997; Tepper & Seldin 1999; Mueller & Scott 2000; Bullani et al. 2001; Thome & Tschopp 2001), the impairment of UPS by cFLIP-L could be involved in the pathological functions of cFLIP-L, such as tumor progression and tumor angiogenesis.

	Experimental procedures

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Plasmids

Human cFLIP-L was cloned into pcDNA-based mammalian expression vectors (Invitrogen) as described previously (Naito et al. 2004). The GFP-degron expression plasmid was constructed by inserting a synthetic oligonucleotide encoding CL1-degron into XhoI and BamHI sites of the pEGFP-C1 (Gilon et al. 1998; Bence et al. 2001). All constructs generated from PCR products were sequenced.

Transfection, immunoprecipitation and immunoblotting

HT-1080 cells and 293T cells were transfected with various plasmid DNAs by lipofection (FuGENE6 (Roche); Lipofectamine 2000 (Invitrogen)). In some cases, cells were treated with benzyloxycarbonyl-valinyl-alanyl-aspartate-fluoromethyl ketone (Z-VAD-fmk) (20 µM) to inhibit the apoptosis induced by cFLIP-L expression. To prepare whole cell lysates, cells were lysed in SDS lysis buffer (0.1 M Tris–HCl at pH 8.0, 10% glycerol, 1% SDS) for 10 min at 100 °C, and cleared by centrifugation at 17 400 g for 10 min. For subcellular fractionation, cells were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagent (PIERCE). The insoluble fractions were lysed in SDS lysis buffer, heated at 100 °C for 10 min and centrifuged at 17 400 g for 10 min.

We used the following antibodies: anti-FLIP (NF-6; Alexis Biochemicals); anti-ubiquitin (2C5), anti-multiubiquitin (FK-2) and polyclonal anti-Myc from MBL; anti-hemagglutinin (anti-HA) and monoclonal anti-Myc from Roche; anti-GFP and anti-Actin from Santa Cruz; anti-RCFP (Clontech); anti-V5 (Invitrogen); and anti-FLAG (M2; Sigma).

Isolation of stable transfectant clones

HT-1080 cells were transfected with pEGFP-degron, pZsProSensor-1 or pEGFP-ubiquitin. At 24 h after transfection, the cells were selected with 500 µg/mL of G-418 for 2 weeks, and the surviving colonies were cloned by limiting dilution. The transfectants were maintained in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 100 µg/mL of kanamycin and 200 µg/mL of G-418 at 37 °C in a humidified atmosphere of 5% CO₂.

Immunofluorescence microscopy

A549 cells were fixed in 4% paraformaldehyde for 30 min at room temperature and treated with 0.1% Triton X-100/3% bovine serum albumin in phosphate-buffered saline for 30 min at 4 °C. Cells were incubated with anti-FLIP (NF6, 1 : 100) as a primary antibody over night at 4 °C and Alexa Fluor 488-conjugated anti-mouse antibody (1 : 1000) as a secondary antibody for 1 h at 4 °C. HT-1080 cells and the cells constitutively expressing GFP-ubiquitin were transfected with Myc-tagged cFLIP-L and fixed as well. HT-1080 cells were incubated with anti-multiubiquitin (FK2, 1 : 200) and anti-Myc (rabbit polyclonal, 1 : 1000) as the primary antibodies over night at 4 °C, then incubated with Alexa Fluor 568-conjugated anti-mouse antibody (1 : 1000) and Alexa Fluor 488-conjugated anti-rabbit antibody (1 : 1000) as the secondary antibodies for 1 h at 4 °C. The GFP-ubiquitin expressing cells were stained with mouse monoclonal anti-Myc (1 : 1000) antibody and Alexa Fluor 568-conjugated anti-mouse antibody (1 : 1000) for 1 h at 4 °C. The cells were observed with an Olympus IX70 microscope equipped with a charge-coupled device camera.

Luciferase assay

Cells in 24-well plates were transfected with a total of 0.5 µg of various combinations of plasmids; 25 ng of reporter plasmid (TOP-TK-Luc, FOP-TK-Luc or 5xHRE/pGL3/VEGF/E1b) (Korinek et al. 1997; Shibata et al. 2000), 2.5 ng of internal control (pRL-TK), 0.25 µg of cFLIP-L expression vector (pcDNA3-Myc) and empty pcDNA3 vector. In some cases, the conditioned medium containing Wnt3a, which was prepared from L cells that had been transfected with the Wnt3a gene as described previously (Shibamoto et al. 1998), was added to the medium. Luciferase activities were measured at 24 h after transfection using the Dual-Luciferase Reporter Assay System (Promega).

RNA interference

pSilencer3.1-H1neo vector (Ambion) designed to express short hairpin RNA (shRNA) against cFLIP-L was constructed as described previously (Naito et al. 2004). The plasmids were transfected using Lipofectamine 2000 (Invitrogen).

	Acknowledgements

 
We thank Drs T. Akiyama for ß-catenin plasmid, H. Clevers for TOP-TK-Luc and FOP-TK-Luc reporter plasmids, and T. Shibata for 5xHRE/pGL3/VEGF/E1b reporter plasmid. We also thank Drs A. Tomida and N. Fujita for helpful discussions. This study was supported by Grants-in Aid for Cancer Research and Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. Pacific Edit reviewed the manuscript prior to submission.

	Footnotes

 
Communicated by: Kohei Miyazono

^* Correspondence: E-mail: mnaito{at}iam.u-tokyo.ac.jp

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Accepted: 25 February 2007

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