C10ORF10/DEPP, a transcriptional target of FOXO3, regulates ROS-sensitivity in human neuroblastoma
© Salcher et al.; licensee BioMed Central Ltd. 2014
Received: 7 February 2014
Accepted: 24 September 2014
Published: 28 September 2014
FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells.
The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor.
We report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells.
The combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.
Decidual Protein induced by Progesterone (DEPP) was originally identified as fasting- and progesterone-induced gene. DEPP is regulated via progesterone in endometrial stromal cells and via insulin levels in adipose tissue and liver and is induced in malignant glioma cells in response to hypoxic stress. It is highly expressed in various tissues including placenta, ovary, kidney, white adipose and liver. The amino acid sequence of DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence, which suggests that DEPP may be imported into peroxisomes. However, the physiological functions of DEPP remain largely unknown[1–3].
In neuroblastoma cells an increased activity of the phosphatidylinositol-3 kinase (PI3K) protein kinase B (PKB/AKT) pathway was reported that contributes to therapy resistance and is associated with phosphorylation and functional inactivation of the transcription factor FOXO3[4, 5]. Phosphorylation of FOXO3 by PKB at distinct amino acids leads to its association with 14-3-3 proteins, resulting in export from the nucleus and as a consequence thereof loss of target gene regulation in neuroblastoma cells. Phosphorylation of FOXO3 by stress-induced kinases such as mammalian Ste20-like kinase (MST1) or c-Jun N-terminal kinase (JNK) in turn stimulates nuclear entry, leading to the activation or repression of target genes that affect growth, cell cycle progression, apoptosis and longevity[7–9].
In neuroblastoma cells, FOXO3 regulates cellular apoptosis by activating the two BH3-only proteins PMAIP1/Noxa and BCL2L11/Bim and sensitizes these tumor cells to chemotherapy-induced cell death by repressing the IAP-family member BIRC5/Survivin. Recently we also demonstrated that DNA-damaging agents activate FOXO3 and thereby cause reactive oxygen species (ROS) formation at the mitochondria due to uncoupling of mitochondrial respiration through the BH3-only protein Bim. ROS are generated as side products of mitochondrial respiration. Under normal conditions, low amounts of ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase, in the mitochondria by superoxide dismutase (SOD2) as well as by members of the sestrin family[6, 9] and by peroxiredoxins which are located in diverse organelles dependent on the cell type. Catalase converts hydrogen peroxide to water and oxygen and is also described as a direct transcriptional target of FOXO transcription factors in various cell types[16, 17]. It is not regulated in neuroblastoma cells. High levels of cellular ROS cause oxidation of proteins, nucleic acids and intracellular membranes thereby impairing cell growth, cellular survival and proliferation[18, 19].
The transcription factor peroxisome proliferator-activated receptor gamma (PPARG) was described to be critical for the regulation of ROS steady state levels, as it directly influences the expression of several ROS-detoxifying enzymes, among them also catalase. The PPARG promoter is repressed by FOXO1 in adipocytes - on the other hand PPARG can also repress the transcriptional activity of FOXO1[21, 22].
Also beta-Catenin, which is regulated via the Wnt-pathway, represses PPARG expression and interacts with both, FOXO3 and PPARG via its TCF/Lef1 binding site (reviewed in). DEPP may affect Wnt-signaling and thereby PPARG expression via its Pro-Pro-Pro-Ser-Pro (PPPSP) motif that has been shown to activate the Wnt-pathway[3, 25].
In the present study we investigated the regulation of DEPP by FOXO3 in human neuroblastoma cells and addressed its effects on cellular ROS household and tumor growth.
FOXO3 regulates DEPP expression on mRNA and protein level in human neuroblastoma cells
All three FOXO binding sites of the DEPP promoter contribute to DEPP-induction by FOXO3
To study whether DEPP is a direct target of FOXO3 in neuroblastoma cells, quantitative RT-PCR analysis of SH-EP/FOXO3 cells treated with 75 nM 4OHT and with 10 μg/ml of the protein biosynthesis inhibitor cycloheximide (CHX) for 2 hours was performed. Treatment with CHX did not prevent the induction of DEPP after FOXO3 activation, which implies that induction of DEPP by FOXO3 does not depend on de novo synthesis of additional proteins, but is due to direct transcriptional regulation (Figure 2a). To further test this hypothesis, a DEPP promoter reporter luciferase assay was performed in SH-EP/FOXO3 cells using a 1116 bp genomic fragment of the promoter cloned upstream of firefly luciferase. The DEPP promoter contains three putative binding sites for FOXO3 (Figure 2b), which were mutated for this experiment. The first binding site for FOXO3 is located at -537 (B1), the second at -179 (B2), and the third at -151 (B3) relative to the start of the DEPP mRNA.
These data demonstrate that FOXO3 activates all three consensus elements in the DEPP promoter and that all three FOXO3 binding sites are important for DEPP regulation by FOXO3 in neuroblastoma cells.
Knockdown of DEPP reduces FOXO3-mediated apoptosis
We recently demonstrated that FOXO3-induced apoptosis is associated with and mediated by a biphasic accumulation of ROS. Thus we analyzed ROS steady state levels in DEPP-knockdown cells and controls at the specific time points by live-cell imaging. Knockdown of DEPP almost completely prevented both, the primary (4 hours and 12 hours) and secondary (16 and 48 hours) ROS increase during FOXO3-activation in SH-EP/FOXO3 and NB15/FOXO3 cells, respectively (Figure 3c). In a previous study we observed that the oxidoreductase p66/SHC1 is strongly phosphorylated at Ser36 during FOXO3-induced ROS accumulation. We therefore performed immunoblot analysis of p66/SHC1 and pSer36-p66/SHC1 protein in SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP cells (three individual clones), which demonstrated that p66/SHC1 was not phosphorylated after FOXO3 activation in DEPP-knockdown cells (Figure 3d). This is consistent with the pronounced effect of DEPP-knockdown on FOXO3-induced ROS accumulation demonstrated by live-cell fluorescence imaging analyses (Figure 3c).
We have previously shown that BCL2L11/Bim induction and BIRC5/Survivin repression are essential for FOXO3-induced ROS accumulation and cell death. Knockdown of DEPP neither prevented Bim induction nor Survivin repression (data not shown), suggesting that knockdown of DEPP does not interfere with the initiation phase of ROS production but might affect cellular ROS detoxification.
DEPP regulates the catalase enzyme activity and thereby the cellular ROS detoxification capacity
DEPP localizes to mitochondria and peroxisomes in neuroblastoma
In this study we demonstrate for the first time that the FOXO3-regulated gene DEPP impairs ROS detoxification via the enzyme catalase and thereby increases the effects of ROS on FOXO3-induced apoptosis in neuroblastoma cells. Using Affymetrix gene expression profiling we identified DEPP as a FOXO3-regulated gene (Figure 1a) in neuroblastoma as well as CEM-C7H2 leukemia cells and found DEPP also induced by FOXO3 at the protein level (Figure 1c). Recently it was reported that DEPP is transcriptionally regulated by FOXO3 in human endothelial cells and that FOXO3 binds to the DEPP promoter at -151 (B1) and -179 (B2) relative to the transcription start. However, we found that the direct induction of DEPP by FOXO3 is critically mediated via a third binding site (B3), which is located -537 relative to the transcription start of the DEPP mRNA in neuronal cells (Figure 2b). Both, luciferase-reporter assays and ChIP analyses demonstrate that FOXO3 binds to the three FOXO-consensus sequences, among them most efficiently to the binding site B3 (Figure 2c).
DEPP expression is inhibited by insulin-growth signaling in neuroblastoma cells (Figure 1d). This is in line with studies describing downregulation of DEPP mRNA in mouse 3T3-L1 adipocytes, rat H4IIE and human HepG2 hepatoma cells as a result of insulin treatment[1, 35]. However, in neuroblastoma cells DEPP-induction by growth factor withdrawal is strongly reduced in the presence of a dominant-negative FOXO mutant suggesting that DEPP regulation by insulin/growth factor signaling almost exclusively relies on FOXO transcription factors (Figure 1d).
Currently there is little known about the physiological function of DEPP as the few studies available focused on the mechanisms of DEPP regulation. Chen and colleagues found a prominent increase of DEPP expression in endothelial cells when cultured under hypoxic conditions. Similar results had been reported before in a malignant glioma cell line. These results may indicate a possible role of DEPP during cellular stress response.
In this paper we demonstrate that the induction of DEPP by FOXO3 contributes to FOXO3-induced cell death as DEPP-knockdown significantly reduced FOXO3-mediated apoptosis (Figure 3b). This death-protective effect of DEPP-knockdown was associated with and possibly also mediated by a marked reduction of FOXO3-induced ROS accumulation (Figure 3c). FOXO3 causes phosphorylation at Ser-36 of p66/SHC1, which correlates with the time point of the second ROS accumulation and apoptosis induction in SH-EP/FOXO3 cells. This phosphorylation of p66/SHC1 was almost completely prevented by DEPP-knockdown (Figure 3d). On the other hand, forced DEPP-overexpression in the absence of FOXO3-induction slightly increased cellular ROS levels, but importantly did not elevate cellular ROS to the level of FOXO3-induced ROS formation. This might explain why DEPP-overexpression did not lead to phosphorylation of p66/SHC1 and apoptosis induction by its own (Figure 4). These combined data suggest that in neuronal cells DEPP may act as a sensitizer for cellular ROS that affects ROS accumulation and/or detoxification.
DEPP contains a PTS2 signal sequence at its N-terminus suggesting that this protein might get imported into peroxisomes, which in turn critically mediate the oxidative stress response via the enzyme catalase. Catalase activity was reduced in response to DEPP overexpression and significantly increased in DEPP-knockdown cells, although catalase was not regulated on protein level (Figure 5a,b). In parallel, the expression of PPARG was strongly upregulated in DEPP-knockdown cells. This transcription factor is critical for the control of cellular ROS levels as it directly regulates several different ROS-detoxifying enzymes, including catalase. PPARG acts as a regulator of peroxisomal proliferation and its upregulation may explain the marked resistance of DEPP-knockdown cells towards H2O2 (Figure 5d). This repression by FOXO3 (Figure 5c) is consistent with earlier studies that demonstrated direct transcriptional repression of the PPARG promoter by FOXO1, which recognizes the same consensus sequence as FOXO3[21, 22]. PPARG on the other hand can also repress FOXO transcriptional activity. Beta-Catenin, which interacts with both, FOXO and PPARG via its TCF/Lef1 binding site, represses PPARG expression (reviewed in). We found beta-Catenin expression up-regulated as a result of DEPP overexpression (Additional file2: Figure S2). One explanation for this phenomenon could be that DEPP stimulates the Wnt-pathway via its PPPSP motif[3, 23, 25]. As beta-Catenin mediates repression of PPARG, this could explain the observed increase in PPARG expression in DEPP-knockdown cells, which in turn increases ROS resistance and further shuts down FOXO3 transcriptional activity. When ectopically expressed, FOXO3 is activated, this circuit is interrupted and PPARG is repressed (Figure 5c), which also opens an avenue for ROS-accumulation during FOXO3-induced cell death. This possible link between DEPP, beta-Catenin and PPARG protein expression will be investigated in a separate project.
Reduced catalase activity results in increased levels of intracellular H2O2 and subsequent cellular damage. Furthermore it was described that the increase in mitochondrial oxidative damage and the decrease in mitochondrial function occurs rapidly following the inhibition of peroxisomal catalase. H2O2 causes oxidative damage throughout the cell and mainly impairs mitochondria, which in turn leads to further ROS accumulation. In particular H2O2, which is the species of ROS that accumulates upon catalase inhibition, freely diffuses across biological membranes, including aquaporin channels present in the mitochondrial membranes. Consistent with the changes in catalase activity, we found that DEPP overexpression increased ROS levels and H2O2-induced apoptosis, whereas DEPP-knockdown cells were more resistant to H2O2 treatment (Figure 5d).
In transient overexpression studies using HEK293T cells DEPP was reported to localize to the nucleus and not to peroxisomes as predicted by the presence of a PTS2 signal in the N-terminus. Using subcellular fractionation (Figure 7a) and live cell confocal imaging with fluorescent peroxisomal and mitochondrial probes (Figure 7b) we demonstrate that in neuroblastoma cells DEPP is a cytoplasmatic protein that localizes in part to peroxisomes and to mitochondrial structures. Co-immunoprecipitation analyses (Figure 7c) indicate that DEPP is targeted to peroxisomes in a PTS2-dependent manner as it interacts with the PEX7 receptor.
As these two cell organelles are the key regulators of cellular stress response and ROS generation/detoxification, DEPP might directly modulate ROS at these organelles.
The above data predict that DEPP may act as a sensitizer for all forms of apoptotic cell death that involve accumulation of ROS as a second messenger. Indeed, this is true for FOXO3-induced apoptosis, where knockdown of DEPP significantly lowered FOXO3-induced cell death (Figure 3b). DEPP overexpression on the other hand increased etoposide-induced apoptosis. Of note, etoposide also activates endogenous DEPP expression, thereby limiting the visible effect of ectopic expression on death sensitivity (Figure 6). Importantly, induction of DEPP by etoposide and death-sensitization by DEPP are in line with our previous work, which demonstrated that etoposide-induced cell death depends on the activation of FOXO3 and the subsequent induction of cellular ROS in neuronal cells.
In neuroblastoma, FOXO3 gets activated as a result of cellular stress response, which leads to cellular ROS formation and upregulation of DEPP expression. The combined data clearly demonstrate for the first time that DEPP regulates cellular ROS levels, reduces catalase enzyme activity and may thereby support or even amplify ROS accumulation during FOXO3-induced apoptosis.
Cell lines, culture conditions, and reagents
The neuroblastoma cell lines STA-NB1, STA-NB3 and STA-NB15 were isolated at the St. Anna Children’s Hospital (Vienna, Austria) and are termed NB1, NB3 and NB15, respectively. SH-EP cells were kindly provided by N. Gross, Lausanne, Switzerland. The acute lymphoblastic leukemia cell line CEM/C7H2, a subclone of the CCRF-CEM cell line and all other cell lines were cultured in RPMI 1640 (Lonza, Basel, Switzerland) containing 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (Gibco BRL, Paisley, GB) at 5% CO2 and 37°C in saturated humidity. PhoenixTM packaging cells for helper-free production of amphotropic retroviruses and HEK293T packaging cells for production of lentiviruses were cultured in DMEM (Lonza, Basel, Switzerland). Cell culture was tested routinely for mycoplasma contamination using the VenorRGeM-mycoplasma detection kit (Minerva Biolabs, Germany). All reagents were purchased from Sigma-Aldrich (Vienna, Austria) unless indicated otherwise. For each experiment, mid-log-phase cultures were seeded in fresh medium.
Retroviral and lentiviral expression vectors
The vectors pLIB-FOXO3(A3)-ER-iresNeo, pQ-tetCMV-SV40-Neo and pQ-tetCMV-EGFP-SV40-Neo have been described previously[5, 41, 42]. For conditional gene expression, the coding region of DEPP was amplified from human cDNA with primers containing appropriate restriction enzyme sites. The fragment was inserted into the MfeI and XhoI sites of the tet-regulated expression plasmid pQ-tetCMV-SV40-Neo generating the plasmid pQ-tetCMV-DEPP-SV40-Neo and into the MfeI and XhoI sites of the EYFP-containing plasmid pQ-tetCMV-EYFP-SV40-Neo (pQ-tetCMV-EYFP-DEPP-SV40-Neo). The lentiviral vectors coding for human DEPP-specific shRNA and the control vector pLKO.1 were obtained from Sigma-Aldrich (Vienna, Austria). pLIB-mycTag-FOXO3-DBD-iresPuro was constructed by inserting the FOXO3-DBD fragment from pSG5-MycTag-FOXO3-DBD into the EcoR1 and Sal1 sites of the pLIB-MCS2-iresPuro plasmid.
Production of retroviruses and lentiviruses for infection of neuroblastoma and leukemia cells
6 × 105 Phoenix™ packaging cells were transfected with 2 μg of retroviral vectors and 1 μg of a plasmid coding for VSV-G protein using Lipofectamine2000 (Invitrogen, Carlsbad, USA). For production of lentiviruses 6.5 × 105 HEK293T cells were transfected with 1.6 μg pLKO.1-shDEPP plasmids coding DEPP-specific shRNAs (Sigma-Aldrich, Vienna, Austria) and the packaging plasmid pCMV 8.91 (kindly provided by D. Trono, EPFL, Lausanne). After 48 hours the virus-containing supernatants were filtered through 0.22 μm syringe filters (Sartorius, Goettingen, Germany) and incubated with the target cells for at least 6 hours. SH-EP/FOXO3 and NB15/FOXO3 cells were infected to generate SH-EP/FOXO3-Ctr, SH-EP/FOXO3-shDEPP (clone-10, -12 and -13), NB15/FOXO3-Ctr and NB15/FOXO3-shDEPP (bulk-selected) cells. pQ-tetCMV-DEPP-SV40-Neo, pQ-tetCMV-EYFP-DEPP-SV40-Neo, and pQ-tetCMV-EGFP-SV40-Neo supernatants were used to generate SH-EP/tetDEPP, SH-EP/tetEYFP-DEPP and SH-EP/tetEGFP cells for doxycycline-inducible DEPP and EGFP expression using the “tet-on” system . pLIB-mycTag-FOXO3-DBD-iresPuro supernatants were used to infect SH-EP cells (SH-EP/FOXO3-DBD) .
Microarray data set generation and analysis
Generation of the Affymetrix microarray data set was performed at the Expression Profiling Unit of the Medical University Innsbruck according to the manufacturer’s protocols. The procedure and protocols have been described elsewhere. The data analysis was performed in R (version). Raw data has been pre-processed using the GCRMA method. Raw and pre-processed data has been deposited at the Gene Expression Omnibus (GEO accession number GSE53046).
Site directed mutagenesis
A luciferase reporter plasmid containing the DEPP promoter (-1116 bp relative to the transcription start site) was purchased from Switchgear Genomics (Menlo Park, USA). The three putative FOXO3 binding sites in the DEPP promoter (named B1, B2 and B3) were mutated by sited directed mutagenesis PCR using circular mutagenesis. The first site is located at -537 to -530 (B1), the second at -179 to -172 (B2), and the third at -151 to -143 (B3) relative to the transcription start (Primers for mutagenesis PCR: B1-fwd: GCTTTCGGAGGATTTGTTTGTCGAC TTGTTCACCAGATAT, B1-rev: ATATCTGGTGAACAAGTCGACAA ACAAATCCTCCGAAAGC, B2-fwd: CTGCCCTGCAGCGTAACTTTT CCCCAGCCTCCTAC, B2-rev: GTAGGAGGCTGGGGAAAAGTTAC GCTGCAGGGCAG, B3-fwd: CAGGCAGAAAACACC CTCCAAGCTGG, B3-rev: CCAGCTTGGAGGGTGTTTTC TGCCTG/ Ta = 58°C, 18 PCR-cycles). Promoter reporter plasmids with mutated FOXO-binding sites B1, B2, B3, B1 + B2 and B1 + B2 + B3 were used for luciferase activity analysis.
Quantitative RT-PCR analysis
To quantify DEPP mRNA levels, we designed “real-time” RT-PCR assays, using GAPDH as reference gene. NB1/FOXO3, NB3/FOXO3, NB15/FOXO3 and SH-EP/FOXO3 cells were cultured in the presence of 100 nM 4OHT for the times indicated to activate the FOXO3(A3)ERtm transgene. Total RNA was prepared from 5 × 106 cells using TRIzolTM Reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA using the Revert H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Huntsville, USA). Quantitative RT-PCR was performed as described previously using DEPP (forward ACTGTCCCTGCTCATCCATTCTC and reverse AGTCATCCAGGCTAGGAGAGGG) and GAPDH-specific oligonucleotides (forward TGTTCGTCATGGGTGTGAACC and reverse GCAGTGATGGCATGGACTGTG). After normalization on GAPDH expression, regulation was calculated between treated and untreated cells.
Immunoblotting and subcellular fractionation
Immunoblot analysis and subcellular fractionation were performed as described previously. The membranes were incubated with primary antibodies specific for DEPP, PEX7, GAPDH (Novus, Littleton, USA), Bim, beta-Catenin (BD Biosciences, Heidelberg, Germany), Catalase (Calbiochem, San Diego, USA), p66/SHC1, phosphorylated pSer36-p66/SHC1 (Abcam, Cambridge, UK), PPARG, Lamin, CoxIV (Cell Signaling,Danvers, USA), GFP (Sigma-Aldrich,Vienna,Austria) and alpha-Tubulin (Oncogene Research Products, Boston,USA).
After incubation with anti-mouse, anti-rat or anti-rabbit horseradish-peroxidase-conjugated secondary antibodies the blots were analyzed by enhanced chemiluminescence substrate (GE-Healthcare, Vienna, Austria) according to the manufacturer’s instructions and detected with an AutoChemiSystem (UVP, Cambridge, GB). Quantification of protein expression was done with the ImageJ 1.48 software, according to the ImageJ User Guide (http://imagej.nih.gov/ij/index.html).
The peroxisomal fraction was isolated by using the peroxisome isolation kit (PEROX1; Sigma-Aldrich, Vienna, Austria), according to the instructions of the manufacturer. Briefly, 4 × 108 SH-EP/tetEYFP-DEPP cells were treated with 200 ng/ml doxy for 24 hours, harvested, resuspended in peroxisome extraction buffer and homogenized in a dounce homogenizer. Next, the cells were centrifuged for 10 minutes at 1000 g. The supernatant represents the “input fraction”. After several centrifugation steps according to the manufacturer’s protocol, the pellet was collected in 1x peroxisome extraction buffer. Isolation of the peroxisomes was done on a density gradient. By a centrifugation step for 1.5 hours at 100.000 g the purified peroxisomes were separated from the mitochondria. To measure the purity of the peroxisomal fraction immunoblot analyses with catalase (peroxisomes) and CoxIV antibodies (mitochondria) were performed.
Co-immunoprecipitation analysis (CoIP)
CoIP was performed with a Pierce Crosslink Magnetic IP/Co-IP kit (Pierce, Rockford, USA) according to the instructions of the manufacturer. Briefly, 5 μg of PEX7 antibody (Novus, Littleton, USA) were covalently cross-linked to 25 μl of A/G magnetic beads. The prepared beads as well as beads without cross-linked antibody (IP Ctr) were incubated with extracts from SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours, washed to remove non-bound material and eluted in a low-pH elution buffer that dissociates bound antigen from the antibody- linked beads. The total protein (TP) and the eluates (IP and IP Ctr) were analyzed via immunoblot.
Chromatin immunoprecipitation assay (ChIP)
ChIP was performed with a Millipore Magna ChIP Kit (Millipore, Darmstadt, Germany) according to the instructions of the manufacturer. Approximately 2 × 107 SH-EP/FOXO3 cells and 20 μl of the protein G beads coupled with 5 μl of anti-FOXO3 antibody (Santa Cruz, Dallas, USA) were used for each preparation. For quantification of FOXO3 binding to the DEPP promoter quantitative real time RT-PCR was performed with primers for the binding sites B1 + B2 (forward AAAACAGCTTGGTGGGCGGG and reverse AACAAGCTTTGGGGCAGGGG) and B3 (forward CTGCTCCTAGGAGAGACACACCCTG and reverse CTGCTACGTTTGCTGTGCTTAGTGC).
Determination of apoptosis by flow cytometry
Apoptosis was measured by staining the cells with propidium-iodide (PI) and forward/sideward scatter analysis using a CytomicsFC-500 Beckman Coulter. 2 × 105 cells were harvested and incubated in 500 μl hypotonic PI solution containing 0.1% Triton X-100 for 4 to 6 hours at 4°C. Stained nuclei in the sub-G1 marker window were considered to represent apoptotic cells.
Luciferase activity assay
To determine direct regulation of the DEPP-promoter by FOXO3, promoter plasmids containing the DEPP-promoter (1116 bp) and mutated variants were transiently transfected into SH-EP/FOXO3 cells using the JetPrime® Reagent (Polyplus, Berkeley, USA) according to the manufacturer’s instructions. Subsequently the cells were cultured in the presence of 100 nM 4OHT for 4 hours to activate the FOXO3 transcription factor. Luciferase activity was measured with a Luciferase Assay System kit (Promega, Fitchburg, USA) according to the manufacturer’s instructions. The reactions were done in duplicates and repeated three times. Luciferase activity was calculated between treated and untreated cells.
Live cell ROS staining
For ROS measurements, cells were grown on LabTek Chamber Slides™ (NalgeNunc International, Rochester, USA) coated with 0.1 mg/ml collagen and incubated with reduced MitoTrackerRed CM-H2XROS (Invitrogen, Carlsbad, CA, USA) for 20 minutes according to the manufacturer’s instructions (final concentration 500 nM). Images were collected with an Axiovert200M microscope equipped with filters for EYFP (exitation: BP500/20, emission: BP535/30) and RFP (excitation: BP546/12, emission: LP590) and a 63x-oil objective (Zeiss, Vienna, Austria).
Live confocal imaging
Cells were grown on LabTek Chamber Slides™ (NalgeNunc International, Rochester, USA) coated with 0.1 mg/ml collagen and incubated for 15 minutes with 30 nM MitoTrackerRed CMX-Ros (Invitrogen, Carlsbad, USA) to stain mitochondria. Peroxisomes were labelled with the CellLight® Peroxisome-RFP vector (Life Technologies, Carlsbad, USA) according to the manufacturer’s instructions. Cells were analyzed by live confocal microscopy using an inverted microscope (Zeiss Observer.Z1; Zeiss, Oberkochen, Germany) in combination with a spinning disc confocal system (UltraVIEW VoX; Perkin Elmer, Waltham, MA, USA). All images were acquired using a 63× oil immersion objective.
Catalase enzyme activity was analyzed with a Catalase Assay Kit (Abcam, Cambridge, UK). Cells were cultured in the presence of 50 nM 4OHT (SH-EP/FOXO3-shDEPP) or 200 ng/ml doxy (SH-EP/tetEGFP, SH-EP/tetDEPP, SH-EP/tetEYFP-DEPP) for the times indicated. 1 × 106 cells were harvested and used for the enzyme assay according to the manufacturer’s instructions. After 30 minutes incubation time the reaction was stopped and the optical density was measured with a Benchmark Microplate Reader (BioRad Laboratories, Munich, Germany). Catalase enzyme activity was calculated between treated and untreated cells.
This work was supported by grants from “Kinderkrebshilfe Tirol und Vorarlberg”, the “Krebshilfe Südtirol”, the “Kinderkrebshilfe Südtirol-Regenbogen” and the “SVP-Frauen-Initiative”. The Tyrolean Cancer Research Institute and this study are supported by the “Tiroler Landeskrankenanstalten Ges.m.b.H. (TILAK)” and the “Tyrolean Cancer Society”.
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