Research Article 1191 FOXO3-induced reactive oxygen species are regulated by BCL2L11 (Bim) and SESN3

Judith Hagenbuchner1,2, Andrey Kuznetsov4, Martin Hermann5, Barbara Hausott6, Petra Obexer1,2,* and Michael J. Ausserlechner1,3,* 1Tyrolean Cancer Research Institute, Innsbruck, Austria 2Department of Pediatrics IV, 3Department of Pediatrics II, 4Cardiac Research Laboratory, Department of Cardiac Surgery, 5KMT Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Center of Operative Medicine, 6Division of Neuroanatomy, Medical University Innsbruck, Innsbruck, Austria *Authors for correspondence ([email protected]; [email protected])

Accepted 6 October 2011 Journal of Cell Science 125, 1191–1203 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.092098

Summary FOXO factors induce and regulate cellular production of reactive oxygen species (ROS). To identify the sequence of molecular events underlying FOXO3 (FKHRL1)-induced apoptosis, we studied the regulation and function of FOXO3 by expressing an ECFP-tagged FOXO3 or a 4OH-tamoxifen (4OHT)-inducible FOXO3–ERtm fusion in SH-EP and STA-NB15 neuronal cells. After knockdown of FOXO3 or expression of a dominant-negative FOXO3 mutant we observed that etoposide- and doxorubicin-induced elevation of cellular ROS depends on FOXO3 activation and induction of its transcriptional target BCL2L11 (Bim). Activation of FOXO3 on its own induced two sequential ROS waves as measured by reduced MitoTrackerRed in live cell microscopy. Induction of Bim by FOXO3 is essential for this phenomenon because Bim knockdown or ectopic expression of BCL2L1 (BclxL) prevented FOXO3-mediated overproduction of ROS and apoptosis. Tetracycline-controlled expression of Bim impaired mitochondrial respiration and caused ROS production, suggesting that FOXO3 induces uncoupling of mitochondrial respiration through Bim. FOXO3 also activated a ROS rescue pathway by inducing the peroxiredoxin SESN3 (Sestrin3), which is responsible for the biphasic ROS accumulation. Knockdown of SESN3 caused an increase of FOXO3-induced ROS and accelerated apoptosis. The combined data clearly demonstrate that FOXO3 activates overproduction of ROS as a consequence of Bim-dependent impairment of mitochondrial respiration in neuronal cells, which leads to apoptosis.

Key words: BCL2 rheostat, Apoptosis, Etoposide, Doxorubicin, Mitochondrial respiration Journal of Cell Science

Introduction Strasser, 2003; Danial and Korsmeyer, 2004; Green and Kroemer, The FoxO subfamily of forkhead transcription factors consists 2004). The family of BCL2 is divided into three of FOXO1 (FKHR), FOXO3 (FKHRL1), FOXO4 (AFX), and subgroups depending on their pro- or anti-apoptotic function and FOXO6 (Calnan and Brunet, 2008; Ho et al., 2008). The regulation are classified by their number of BH domains. The pro-survival of these transcription factors is under the control of the BCL2, BCL2L1 (BclxL), MCL1, BCL2L2 (Bcl-w) and A1 share phosphatidylinositol-3-kinase (PI3K) and protein kinase B (PKB) four BH domains, whereas the ‘multi-domain’ proteins BAX and signaling pathway (Arden and Biggs, 2002) and depends on the BAK1 (Bak) contain only three BH domains and are pro-apoptotic. availability of growth factors. Phosphorylation of FOXOs by PKB The third group are the pro-apoptotic ‘BH3-only’ proteins e.g. on distinct threonine and serine residues causes their export into the PMAIP1 (Noxa), BCL2L11 (Bim), BID and BBC3 (Puma), which cytoplasm. Growth factor withdrawal shuts down PI3K–PKB contain only one BH domain. The ratio of pro- and anti-apoptotic signaling and consequently leads to nuclear accumulation and proteins determines cell fate at the level of mitochondria. Specific activation of FOXO proteins. In addition to survival signaling, ‘BH3-only’ proteins, such as BID or Bim bind all pro-survival FOXO proteins are also subject to a complex regulation by stress BCL2 proteins and thereby displace BAX and Bak from anti- signaling that overrides inactivation through PKB. Jun-N-terminal apoptotic BCL2 family members (Danial and Korsmeyer, 2004). kinase (JNK) (Essers et al., 2004) and mammalian-Ste20-like This results in activation and oligomerization of BAX and Bak, kinase-1 (MST1) (Lehtinen et al., 2006) activate FOXO proteins in which further leads to the permeabilization of the outer response to oxidative stress even in the presence of growth factor mitochondrial membrane (MOMP), the release of cytochrome c signaling. FOXO3 targets are involved in the regulation of and subsequent activation of the caspase cascade. apoptosis, the cell cycle and in the defense against oxidative One apoptotic stimulus of the intrinsic death pathway is the stress (Calnan and Brunet, 2008; Ho et al., 2008). accumulation of reactive oxygen species (ROS) at the 2 Apoptosis can be induced either by the extrinsic pathway, where mitochondria. Hydrogen peroxide (H2O2), superoxide (O2 )or death ligands bind on cell surface death receptors, or by the hydroxyl radicals (OH) are short-lived molecules (Scherz-Shouval intrinsic pathway, which is controlled by the balance of pro- and and Elazar, 2007) that are generated as by-products of anti-apoptotic BCL2 proteins at the mitochondria (Coultas and mitochondrial respiration (Kim et al., 2007). They might 1192 Journal of Cell Science 125 (5)

function as specific messengers by modifying target molecules or doxorubicin and etoposide (Obexer et al., 2009). To investigate by changing the intracellular redox state (Thannickal and Fanburg, whether DNA-damaging drugs affect steady state expression of 2000). High levels of ROS can also damage proteins, nucleic acids FOXO3, we analyzed FOXO3 protein levels after treatment with and intracellular membranes, leading to oxidative stress and etoposide and doxorubicin in neuroblastoma cells. We found that, impairing cellular functions (Hasegawa et al., 2008; Menon et al., upon treatment, endogenous FOXO3 protein levels increased in 2007). Under normal conditions, low amounts of ROS levels are SH-EP (Fig. 1C, left panels) and STA-NB15 cells (Fig. 1C, right eliminated by glutathione metabolism, scavenging enzymes such panels) after 2 and 4 hours. To determine whether the observed as SOD2 (MnSOD) and CAT (), peroxisomes or by accumulation of FOXO3 correlates with its activation, members of the sestrin family (Kim et al., 2007; Kopnin et al., we measured the protein levels of its apoptosis-inducing 2007; Scherz-Shouval and Elazar, 2007). The mitochondrial downstream targets Bim and Noxa. Both pro-apoptotic BH3- 2 enzyme MnSOD converts O2 to H2O2, which is further only proteins were rapidly induced within 2 to 4 hours of drug dismutated to H2OandO2 by catalase. MnSOD and catalase are treatment in SH-EP and STA-NB15 cells. Interestingly, Bim was transcriptional targets of FOXO3 and NF-kB (Chiribau et al., phosphorylated at Ser69, a recognition site for JNK, which 2008; Liu et al., 2005; Storz et al., 2005). SESN3 (Sestrin3), a stabilizes Bim during cellular stress in neuronal cells (Becker member of the sestrin family, is also regulated by FOXO3 et al., 2004). Phosphorylation by JNK is also thought to release (Nogueira et al., 2008). Insufficient defense results in Bim from dynein light chain-1 and to contribute to its activation high levels of ROS, which severely damage the cell and cause (Lei and Davis, 2003). The stress-induced kinases JNK and changes in cellular ATP and Ca2+ levels. This eventually leads to MST1 (Essers et al., 2004; Lehtinen et al., 2006) or the the release of cytochrome c and the induction of apoptosis (Satoh interaction with ataxia telangiectasia mutated (ATM) (Tsai et al., 2004; Kuznetsov et al., 2008a). et al., 2008) might trigger nuclear accumulation of FOXO3 High stage neuroblastoma tumor cells frequently show during the DNA-damage response. To investigate whether increased phosphorylation of PKB on Ser473 and Thr308, FOXO3 also translocates to the nucleus in etoposide- and which leads to hyperactivation of PKB signaling, inactivation doxorubicin-treated neuronal cells we used SH-EP cells designed of FOXO3, the most prevalent FOXO in to express an ECFP–FOXO3 fusion protein (Obexer et al., 2009) neuronal cells, and correlates with a poor prognosis (Opel et al., and analyzed the subcellular localization of the fusion protein by 2007). We have previously shown that activation of FOXO3 live-cell fluorescence imaging. In untreated cells, the ECFP– induces apoptosis in neuronal cells by induction of the pro- FOXO3 fusion protein was mainly localized in the cytoplasm. apoptotic proteins Bim and Noxa and sensitizes to chemotherapy Consistent with earlier studies in non-neuronal cells (Essers et al., by repression of the anti-apoptotic protein BIRC5 (survivin) 2004), H2O2 caused rapid nuclear accumulation of FOXO3 (Obexer et al., 2007; Obexer et al., 2009). However, although within 20 to 40 minutes in neuroblastoma cells (supplementary FOXO3-mediated induction of Bim and Noxa and repression of material Fig. S1A). Etoposide induced nuclear localization of survivin occur within 8 hours, apoptosis execution is delayed and FOXO3 in 72% of the cells within 90 minutes and doxorubicin starts between 24 and 48 hours. Therefore the purpose of the activated FOXO3 in 62% of all cells within 2 hours (Fig. 1D). study was to dissect the molecular mechanisms and the precise Although H2O2 is an efficient activator of FOXO3, in drug-

Journal of Cell Science sequence of events that lead to activation of FOXO3 by treated neuroblastoma cells nuclear accumulation of ECFP– chemotherapeutic agents, and to apoptosis by FOXO3 in FOXO3 preceded ROS accumulation. We therefore addressed neuronal tumor cells. In this study, we focused on the early whether activation of FOXO3 occurs downstream or upstream of events at the mitochondria after FOXO3 activation and ROS production during chemotherapy and pretreated SH-EP cells uncovered the downstream targets of FOXO3 that contribute to stably transfected with ECFP–FOXO3 with the antioxidant N- ROS formation and neuronal cell death. actetyl-L-cysteine (NAC) for 1 hour before they were incubated with etoposide and doxorubicin. Although NAC treatment Results diminished ROS levels (Fig. 3C), the inhibition of ROS Etoposide and doxorubicin activate FOXO3, induce the production did not prevent shuttling of FOXO3 into the nucleus expression of the BH3-only proteins Bim and Noxa, and (Fig. 1D), implying that activation of FOXO3 is not a consequence cause a transitory production of ROS of ROS accumulation. To verify whether the effects of etoposide Etoposide treatment is associated with the production of ROS, the and doxorubicin on ROS generation and increase in Bim depend induction of the prosurvival antioxidant enzyme superoxide on FOXO3 activation, SH-EP and STA-NB15 cells were infected dismutase and the activation of the proapoptotic BH3-only with lentiviruses for FOXO3-specific shRNA expression. After protein Bim through FOXO3 in breast cancer cells (Liu et al., infection, bulk-selected NB15 cells and individual clones from 2005). To study whether ROS also lead to FOXO3 activation SH-EP cells were subjected to immunoblotting (supplementary during chemotherapy-induced apoptosis in neuronal cells, the material Fig. S1B) and analyzed for ROS production and the neuronal tumor cell lines SH-EP and STA-NB15 were treated expression of the FOXO3 target Bim after etoposide and with etoposide and doxorubicin and ROS production was doxorubicin treatment. Live-cell fluorescence images of measured by reduced MitoTrackerRed (CM-H2XROS). Both etoposide-treated SH-EP/shFOXO3-17-clone7 (Fig. 2A) and chemotherapeutic agents caused a transitory increase of ROS at NB15/shFOXO3 cells (Fig. 2B) showed no significant ROS the mitochondria. In both cell lines, ROS reached a maximum accumulation compared with the controls, SH-EP/shCtr and after 2 hours of etoposide treatment and declined after 4 hours. NB15/shCtr, respectively. Next, we determined the Bim Doxorubicin-induced ROS accumulated later and reached a expression level in SH-EP/shFOXO3-17-clone7 cells expressing maximum after 6 hours treatment (Fig. 1A,B). We have shown FOXO shRNA, bulk-selected NB15/shFOXO3-17 cells and before that low-dose activation of an ectopic FOXO3 sensitizes control cells, respectively. Knockdown of FOXO3 completely neuronal cells to death by DNA-damaging agents such as prevented induction of Bim by etoposide for up to 16 hours in both FOXO3 induces ROS during apoptosis 1193 Journal of Cell Science

Fig. 1. Chemotherapeutic agents activate FOXO3 and induce ROS in neuronal cells. Accumulation of ROS was detected using MitoTracker Red CM-

H2XROS by live-cell imaging of SH-EP (A) or STA-NB15 (B) neuroblastoma cells treated with doxorubicin or etoposide for the indicated time points. (C) SH- EP cells were treated with 20 mg/ml etoposide or 0.25 mg/ml doxorubicin (left) and STA-NB15 cells were treated with 10 mg/ml etoposide or 0.5 mg/ml doxorubicin for 0, 2 and 4 hours (right). Cell lysates were subjected to immunoblot analyses using antibodies against FOXO3, BimEL, phosphorylated Bim(Ser69) and Noxa. GAPDH was used as loading control. (D) Translocation of FOXO3 was analyzed in SH-EP cells expressing an ECFP–FOXO3 fusion protein. Cells were treated with etoposide (20 mg/ml) or doxorubicin (0.25 mg/ml) or pre-incubated (1 hour) with 7.5 mM N-acetyl-L-cysteine and analyzed by live-cell fluorescence microscopy in an Axiovert200M fluorescence microscope. For quantification .50 cells were counted and the distribution of cells with nuclear or cytoplasmic localization of FOXO3 was plotted as the mean of four individual experiments (n54). 1194 Journal of Cell Science 125 (5) Journal of Cell Science

Fig. 2. FOXO3 activation is necessary for ROS accumulation. ROS accumulation was detected using MitoTracker Red CM-H2XROS by live-cell imaging of SH-EP/shCtr, SH-EP/shFOXO3-17-clone7 and SH-EP/FOXO3-DBD cells treated with 20 mg/ml etoposide (2 hours) or 0.25 mg/ml doxorubicin (6 hours) (A) or in NB15/shCtr, NB15/shFOXO3-17 and NB15/FOXO3-DBD cells treated with 10 mg/ml etoposide or 0.5 mg/ml doxorubicin (B). Immunoblot analysis of Bim expression in SH-EP/shCtr and SH-EP shFOXO3-17-clone7 cells treated with 20 mg/ml etoposide (C) or NB15/shCtr and NB15/shFOXO3-17 cells treated with 10 mg/ml etoposide (D), respectively, for the times indicated. a-Tubulin served as a loading control. Densitometry was performed using Labworks software (UVP) and intensities were calculated as percent of untreated controls normalized to a-tubulin.

neuroblastoma cell lines (Fig. 2C,D). In a parallel experiment, we FOXO3 induces biphasic ROS accumulation, which infected SH-EP and STA-NB15 cells with a retrovirus coding for a critically contributes to FOXO3-induced cell death dominant-negative FOXO3 mutant (FOXO3-DBD). Ectopic FOXO transcription factors are regulators of cell death but are also expression of the -tagged FOXO3-DBD construct was reported to protect cells against ROS. These contrasting abilities verified by immunoblotting (supplementary material Fig. S1B). might depend on post-translational modifications and cell type. To ROS accumulation during treatment with etoposide and assess how FOXO3 contributes to ROS production in neuronal cells, doxorubicin was prevented by FOXO3-DBD in both cell lines we used SH-EP and NB15 cells that express a conditional, PKB- (Fig. 2A,B). These data suggest that in contrast to previous phosphorylation-independent FOXO3(A3)-ERtm allele, which can findings, the chemotherapeutic agents etoposide and doxorubicin be activated by 4OH-tamoxifen (4OHT) (Obexer et al., 2007). SH- activate FOXO3, increase the expression of FOXO3 target EP/FOXO3 cells or mock-infected controls were treated with and subsequently lead to ROS accumulation in neuronal cells. 100 nM 4OHT for 18 hours or with H2O2 as a positive control. FOXO3 induces ROS during apoptosis 1195

Both, H2O2 and FOXO3 activation strongly increased ROS ROS. Nuclei of cells were stained with Hoechst 33342 dye and staining as measured by reduced MitotrackerRed CM-H2XROS analyzed by fluorescence microscopy. NAC efficiently prevented (supplementary material Fig. S1C). To specify the time points of the first ROS burst at 4 hours of 4OHT treatment in SH-EP/FOXO3 ROS accumulation, we performed time course analysis of 4OHT- cells as measured by CM-H2XROS (Fig. 3C). As shown in Fig. 3D, treated SH-EP/FOXO3 and NB15/FOXO3 cells (Fig. 3A,B). combined treatment with NAC and 4OHT delayed the onset of FOXO3 activation resulted in a highly significant increase of ROS apoptosis, because only 9.9% of cells showed fragmented nuclei after 16 to 18 hours (P,0.0001) in SH-EP/FOXO3 cells, which compared with 37.5% in 4OHT-treated SH-EP/FOXO3 cells. This marks the onset of apoptosis in these cells. In NB15/FOXO3 cells, indicates that cellular ROS levels critically contribute to FOXO3- the climax of ROS accumulation was observed 48 hours after induced programmed cell death in neuronal cells and implies that FOXO3 activation, again coinciding with the onset of programmed FOXO3 triggers a primary ROS increase that induces a secondary, cell death (Obexer et al., 2007). In both cell lines, FOXO3 activation apoptosis-inducing ROS burst. induced a transient highly significant accumulation of ROS, which peaked at 4 hours in SH-EP cells and at 12 hours in NB15 cells, Induction of SESN3 by FOXO3 causes a transitory decline respectively, with significantly decreased ROS levels at the in ROS production and delays FOXO3-induced cell death following time points (Fig. 3B). To investigate the relevance of of neuronal cells. the first ROS burst for cell death induction by FOXO3, SH-EP/ The enzymes MnSOD and catalase were reported to be FOXO3 FOXO3 cells were treated with 4OHT for 16 hours alone or after target genes that critically participate in ROS detoxification pre-incubation with the ROS-inhibitor NAC (1 hour) to scavenge (Chiribau et al., 2008; Kops et al., 2002; Yalcin et al., 2008). Journal of Cell Science

Fig. 3. FOXO3 induces ROS-dependent apoptosis in neuroblastoma cells. (A) Accumulation of ROS was analyzed in SH-EP/FOXO3 or NB15/FOXO3 cells treated with 100 nM 4OHT for 0–20 hours or 0–72 hours, respectively. (B) Relative ROS accumulation was quantified using the Axiovert200M fluorescence microscope software. Each panel represents the mean 6 s.e.m. of 15 to 30 individual cells. Statistical analysis was performed using unpaired Student’s t-test. ***P,0.0001 for ROS intensity compared with untreated controls; ###P,0.0001 for comparison between first ROS climax and subsequent time point. (C)Asa positive control, ROS accumulation was measured in SH-EP/FOXO3 cells treated for 4 hours with 100 nM 4OHT alone or after pre-incubation (1 hour) with 7.5 mM NAC. (D) SH-EP/FOXO3 cells were treated for 16 hours with 100 nM 4OHT alone or pre-incubated for 1 hour with 7.5 mM NAC and analyzed by live- cell fluorescence microscopy in an Axiovert200M fluorescence microscope. Magnified areas indicate fragmented cell nuclei stained with Hoechst 33342 (100 ng/ml). For quantification .100 cells were counted and plotted as the mean of three individual experiments (n53). 1196 Journal of Cell Science 125 (5)

MnSOD converts superoxide into hydrogen peroxide, which is that these two enzymes are not responsible for cellular ROS further converted by catalase to oxygen and water. To determine fluctuations. Consistently, ectopic MnSOD expression also did the possible involvement of these enzymes in the biphasic not prevent ROS induction by FOXO3 (supplementary material ROS accumulation during FOXO3 activation we determined Fig. S1D). expression of MnSOD and catalase protein by immunoblotting. Because MnSOD and catalase were not critical for biphasic No significant induction of these enzymes by FOXO3 was ROS accumulation during FOXO3 activation, we analyzed the observed in SH-EP and STA-NB15 cells (Fig. 4A), indicating regulation of SESN3, a member of the sestrin family, which was Journal of Cell Science

Fig. 4. SESN3, but not MnSOD or catalase, regulates ROS detoxification after FOXO3 activation. (A) Steady state expression of MnSOD and catalase was analyzed by immunoblot analyses in SH-EP/FOXO3 (left) and NB15/FOXO3 cells (right) after incubation with 100 nM 4OHT for 0, 4, 8, 16 and 24 hours, respectively. (B) SESN3 expression was determined by quantitative RT-PCR or immunoblot in SH-EP/FOXO3 (top) and NB15/FOXO3 (bottom) cells treated with 100 nM 4OHT for the times indicated. Bars represent mean values of three independent experiments, each performed in triplicate, *P,0.05; **P,0.01 compared with the untreated control, unpaired Student’s t-test. (C) Knockdown of SESN3 in individual clones was confirmed by RT-PCR. ROS accumulation was detected in SH-EP/FOXO3-shCtr, SH-EP/FOXO3-shSESN3-28-clone12 and SH-EP/FOXO3-shSESN3-46-clone16 cells after treatment with 75 nM 4OHT for 0,

2, 4 and 6 hours using reduced MitoTrackerRed CM-H2XROS. (D) SH-EP/FOXO3-shCtr, SH-EP/FOXO3-shSESN3-28-clone12 and SH-EP/FOXO3-shSESN3- 46-clone16 cells were treated for 24 hours with 75 nM 4OHT and subjected to apoptosis detection by PI-FACS analyses. Shown are means 6 s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, **P50.003; ***P50.0005. FOXO3 induces ROS during apoptosis 1197

recently shown to be regulated by FOXO3 (Nogueira et al., 2008) 36 hours in STA-NB15 cells (Obexer et al., 2007), neutralization and which we identified as a FOXO3 target by Affymetrix of the pro-survival BclxL occurs within 6 hours of FOXO3 expression profiling in neuroblastoma cells (data not shown). As activation and thereby precedes the first accumulation of ROS in shown in Fig. 4B, SESN3 mRNA was strongly induced in SH- STA-NB15 cells. EP/FOXO3 cells (about 50-fold after 6 hours, P50.004) and to a lesser but significant extent in NB15/FOXO3 cells. In parallel, Bim and BclxL critically control FOXO3-induced ROS SESN3 protein levels also increased continuously after FOXO3 accumulation and mitochondrial respiration activation in both cell lines (Fig. 4B, right panels). This induction In line with the hypothesis that ROS are triggered by the of SESN3 correlates with the first decrease of ROS after 4 hours disturbance of the BCL2 rheostat, we analyzed whether BclxL, in SH-EP/FOXO3 and after 12 hours in NB15/FOXO3 cells. To Bim or Noxa are crucially involved in the first ROS induction by investigate whether SESN3 participates in ROS detoxification, FOXO3 in neuronal cells. To address this, we used SH-EP/ SH-EP/FOXO3 and NB15/FOXO3 cells were infected with FOXO3 and NB15/FOXO3 cells that either overexpress BclxL or lentiviruses coding for different SESN3-specific shRNAs. prevent induction of Bim or Noxa by shRNA-mediated gene Knockdown of SESN3 in bulk-selected NB15/FOXO3- knockdown (Obexer et al., 2007). As shown in Fig. 6A, ectopic shSESN3 cells (supplementary material Fig. S2A) and in expression of BclxL or knockdown of Bim completely prevented single clones of SH-EP/FOXO3-shSESN3 cells was verified ROS accumulation after 2 and 4 hours of FOXO3 activation by quantitative RT-PCR (Fig. 4C). Knockdown of SESN3 (P,0.0001), whereas knockdown of Noxa had an attenuating prevented the ROS decline after 6 hours of FOXO3 activation effect only at the 4 hour time-point. Similar results were achieved (Fig. 4C) and increased FOXO3-induced apoptosis from 12% in in NB15/FOXO3 cells, i.e. ectopic BclxL or knockdown of Bim mock-infected control cells to 29% and 24% in FOXO3- markedly reduced ROS accumulation after 12 hours of FOXO3 shSESN3-28-clone12 and FOXO3-shSESN3-46-clone16 SH-EP activation (Fig. 6B). As in SH-EP cells, knockdown of Noxa in cells, respectively, as determined by propidium iodide (PI) FACS NB15 cells reduced the climax of ROS generation at 12 hours, analyses (Fig. 4D). In NB15/FOXO3-shSESN3-46 cells, SESN3 but did not prevent the ROS peak. These results indicate that the knockdown increased apoptosis from 19% to 37% after 48 hours initial ROS production is triggered downstream of Bim and of 4OHT treatment (supplementary material Fig. S2B). This BclxL and that changes in the expression of these two BCL2 implies that FOXO3-induced upregulation of SESN3 scavenges proteins might impair mitochondrial function. To address ROS after a primary ROS accumulation and thereby defines a whether these events are accompanied by changes in cell-protective, apoptosis-delaying ROS threshold. mitochondrial function, we measured endogenous mitochondrial respiration during FOXO3 activation using high-resolution Bim and Noxa neutralize BclxL at the mitochondria and respirometry. Within 4 hours of 4OHT treatment, mitochondrial regulate the interaction of BAX and Bak with BclxL early respiration decreased (Fig. 6C), which correlated with ROS during FOXO3 activation accumulation (Fig. 3A and Fig. 6A) and induction of Bim by We have shown before that FOXO3 controls the expression of the FOXO3 (supplementary material Fig. S3B). Ectopic BclxL pro-apoptotic BH3-only proteins Bim and Noxa, which cause loss significantly prevented the decline of mitochondrial respiration

Journal of Cell Science of mitochondrial membrane potential and eventually lead to (P,0.05), suggesting that the balance between pro- and anti- cytochrome c release in neuronal cells (Obexer et al., 2007). apoptotic BCL2 proteins directly affects the mitochondrial Because accumulation of ROS can be triggered by opening of the activity during FOXO3 activation (Fig. 6A,B). mitochondrial permeability transition pore (MPT) (Zorov et al., Bim is a key regulator of FOXO3-induced apoptosis of 2006), we determined the steady state expression of anti- neuronal cells (Obexer et al., 2007) and its knockdown prevents apoptotic BCL2 proteins after FOXO3 activation. BclxL FOXO3-induced ROS accumulation (Fig. 6A,B). To study protein levels decreased within 8 hours in SH-EP/FOXO3 cells whether Bim causes cellular ROS on its own, we conditionally and declined in NB15/FOXO3 cells after 24 hours of 4OHT expressed Bim in a tetracycline-regulated manner (Fig. 7A) treatment (Fig. 5A), whereas BCL2 was not regulated and MCL1 (Ausserlechner et al., 2005; Hagenbuchner et al., 2010). was slightly induced (supplementary material Fig. S3A). Because Induction of Bim rapidly induced ROS within 2 hours ectopic expression of BclxL significantly prevented FOXO3- (Fig. 7B) and lowered mitochondrial respiration from 100% in induced cell death for up to 72 hours (Fig. 5B), we assessed the mock-infected control cells to 67.664.6% and 49.869.4% after 2 interaction of BclxL with the pro-apoptotic BCL2 proteins Noxa, and 4 hours (P,0.01), respectively. From the combined data, we Bim, BAX and Bak in mitochondria in NB15/FOXO3 cells conclude that induction of Bim by FOXO3 triggers FOXO3- before ROS accumulation could be detected (Fig. 5C). The induced ROS accumulation during DNA-damaging drug-induced BH3-only proteins Bim and Noxa rapidly increased in the cell death in neuronal cells. mitochondrial fraction and co-purified with BclxL within 3 to 6 hours of FOXO3 activation. We recently provided evidence Discussion that Noxa binds to and neutralizes BclxL during proteasome- In this study we demonstrate for the first time that the activation inhibitor-induced apoptosis, suggesting that in neuronal cells, of the transcription factor FOXO3 leads to biphasic ROS BclxL is a preferential pro-survival binding partner of Noxa accumulation and we provide evidence that ROS are critical (Hagenbuchner et al., 2010). Whereas in untreated cells, neither mediators of FOXO3-induced cell death in neuronal cells. Others BAX nor Bak associated with BclxL, the activation of FOXO3 have shown that etoposide activates FOXO3 as a consequence of induced a transitory interaction of Bak and BAX with BclxL at intracellular ROS accumulation in breast cancer cells (Liu et al., 3 hours. At 6 hours, however, BclxL was mainly associated with 2005). However, in neuronal tumor cells, in primary neurons and Noxa and Bim and to a lesser extent with BAX. This suggests in NGF-differentiated PC12 cells (supplementary material Fig. that, although nuclear fragmentation is first detectable after 24 to S4), we demonstrate that etoposide- and doxorubicin-induced cell 1198 Journal of Cell Science 125 (5)

Fig. 5. Ectopic BclxL prevents FOXO3- induced apoptosis by inactivating pro- apoptotic BCL2 family members. (A) BclxL and GAPDH expression was analyzed in SH- EP/FOXO3 (left) and NB15/FOXO3 cells (right) after incubation with 100 nM 4OHT for 0, 4, 8, 16, 24 and 48 hours. (B) SH-EP FOXO3 (left) and NB15/FOXO3 cells (right) were retrovirally infected with a vector coding for BclxL. The ectopic expression was verified by immunoblot analysis. Cells were treated with 100 nM 4OHT for the times indicated and subjected to PI-FACS analyses. Shown are means 6 s.e.m. of three independent experiments; the difference between controls and BclxL transgenic cells at each time point was assessed using unpaired Student’s t-test (*P,0.05; ***P,0.0001). (C) NB15/FOXO3 cells were treated for 3 and 6 hours with 100 nM 4OHT, lysed and subjected to co- immunopurification using anti-BclxL as precipitating antibody. Mitochondrial immunoprecipitates were analyzed for the

Journal of Cell Science presence of BclxL (precipitation control), Bim, Noxa, Bak, BAX, and CoxIV (purification control) by immunoblot.

death depends on the activation of FOXO3 and the subsequent Ser69 (Fig. 1C). In neurons, Bim Ser69 can be phosphorylated by induction of cellular ROS. Chemotherapeutic agents cause JNK or by the pro-survival kinases ERK1/2 leading either to Bim nuclear accumulation of FOXO3 before and independent of activation (Becker et al., 2004) or, in the case of ERK1/2 to ROS accumulation, as evidenced by live-cell fluorescence proteasomal degradation. Because the activation status of JNK imaging (Fig. 1, Fig. 2A,B, supplementary material Fig. was not altered during drug treatment as measured by phospho- S4B,D). Although SH-EP and STA-NB15 cells differ in their specific antibodies directed against Thr183 and Tyr185 status (Hagenbuchner et al., 2010), FOXO3 was activated by (supplementary material Fig. S4E), and inhibition of JNK did etoposide and doxorubicin in both cell lines (Fig. 1C,D), not prevent induction of Bim, it is possible that Ser69 suggesting that induction of FOXO3 does not depend on a phosphorylation results from counter-regulatory activation of functional p53 pathway. Shuttling of FOXO3 might be triggered ERK1/2. Tsai and colleagues demonstrated that during DNA by JNK or by the downstream kinase MST1, which both induce damage the ATM kinase physically interacts with FOXO3 and nuclear accumulation of FOXO transcription factors during can recruit FOXO3 to the nucleus (Tsai et al., 2008). In neuronal cellular stress (Essers et al., 2004; Lehtinen et al., 2006; Bi et al., cells, both etoposide and doxorubicin induce auto- 2010). The tyrosine kinase c-Abl which is also a transducer of phosphorylation of the ATM kinase at Ser1981, which signals for oxidative stress and DNA-damage acts upstream of indicates ATM activation. The inhibition of ATM during drug MST1 and phosphorylates MST1 at Tyr433 causing stabilization, treatment also blocks FOXO3-dependent induction of Bim (our activation and enhanced interaction of MST1 with FOXO3. unpublished results). We therefore believe that ATM contributes Similarly to our experiments, the authors provided evidence that to FOXO3 activation during drug treatment of neuronal cells. activation of FOXO3 by the c-Abl–MST1 pathway promotes When investigating the relationship between FOXO3, cellular apoptosis in mammalian neurons (Xiao et al., 2011). Bim as the ROS levels and cell death in neuronal cells we found that FOXO3 critical mediator of FOXO3-induced ROS was not only induced on its own increased intracellular ROS and induced two at the protein level, but also showed increased phosphorylation at consecutive waves of ROS accumulation (Fig. 3A,B). To FOXO3 induces ROS during apoptosis 1199 Journal of Cell Science

Fig. 6. BclxL and Bim are critical regulators of mitochondrial respiration and ROS accumulation by FOXO3. (A) SH-EP/FOXO3-Ctr, SH-EP/FOXO3- BclxL, SH-EP/FOXO3-shBim-clone1 and SH-EP/FOXO3-shNoxa-clone3 cells were treated with 100 nM 4OHT for 2, 4 and 6 hours and analyzed for ROS accumulation. (B) ROS accumulation was determined in NB15/FOXO3-Ctr, NB15/FOXO3-BclxL, NB15/FOXO3-shBim and NB15/FOXO3-shNoxa cells after incubation with 100 nM 4OHT for 0, 10, 12 and 14 hours. Bottom panels in A and B show relative ROS accumulation quantified using the Axiovert200M fluorescence microscope software. Bars represent means 6 s.e.m. of ROS intensity of 15–30 cells. Statistical analysis was performed using unpaired Student’s t-test. ***P,0.0001 untreated cells compared with 4OHT-treated cells; ###P,0.0001 comparison between identical time point of control cells and BclxL- or shBim-expressing cells. (C) Mitochondrial respiration was measured in SH-EP/FOXO3 and SH-EP/FOXO3-BclxL cells after treatment with 100 nM 4OHT for 0 and 4 hours (mean values of three independent experiments 6 s.d.; *P,0.05 compared with BclxL 4 h).

further investigate the underlying molecular reasons for this 2005; Tan et al., 2008). These enzymes were not regulated by biphasic ROS burst, we evaluated the expression of the FOXO3 in the examined neuronal cells and therefore cannot ROS-detoxifying enzymes MnSOD and catalase, both of which contribute to the fluctuation of ROS levels during FOXO3 are reported to be downstream targets of FOXO3 (Liu et al., activation (Fig. 4A). However, we identified SESN3 as a strongly 1200 Journal of Cell Science 125 (5)

stress, they cannot be reduced and thereby regenerated to the active form by thioredoxin. This conversion of overoxidized peroxiredoxins to active scavengers requires ATP-dependent reductases of the sestrin family (Budanov et al., 2004). As a result of this function, sestrins are crucial for keeping ROS-scavenging peroxiredoxins active. Because knockdown of SESN3 prevented the transitory decline in ROS during FOXO3 activation and accelerated FOXO3-induced cell death in these neuronal cells (Fig. 4C,D, supplementary material Fig. S2A,B), FOXO3, through SESN3, seems to activate both ROS production and a ROS-detoxifying pathway. Zorov and colleagues showed that mitochondrial ROS trigger a secondary enhanced ROS production leading to significant mitochondrial and cellular injury (Zorov et al., 2006). Mitochondrial stress or the opening of channels in the outer or inner mitochondrial membrane might result in a collapse of the mitochondrial membrane potential (DY) and could transiently increase ROS levels by the electron transfer chain. Because reduced respiratory activity (Fig. 6C) and increase in ROS are observed during initial FOXO3 activation, we hypothesized that FOXO3-induced changes in expression and activation of BCL2 proteins cause a transient opening of the MPT pore. FOXO3 induces the pro-apoptotic proteins Noxa and Bim and leads to release of cytochrome c in neuroblastoma cells (Obexer et al., 2007). The induction of Bim and Noxa within the first 6 hours correlates with a transitory association of BAX and Bak with BclxL at the mitochondria (Fig. 5C). This dynamic interaction might be due to Noxa- and Bim-induced redistribution of BAX and Bak between different pro-survival BCL2 proteins (Willis et al., 2005). Between 3 and 6 hours, however, BAX and Bak are partially displaced from BclxL, allowing them to activate MPT formation. Importantly, conditional expression of Bim triggers ROS accumulation (Fig. 7B) and remarkably reduces Fig. 7. Bim induces ROS accumulation. (A) SH-EP/tetCtr and SH-EP/Bim mitochondrial respiration (Fig. 7C). Moreover, FOXO3-induced

Journal of Cell Science cells were treated with 200 ng/ml doxycycline (doxy) for 0, 2, 4 and 8 hours ROS production is prevented by knockdown of Bim or ectopic and subjected to immunoblot analysis for Bim expression. GAPDH serves as expression of BclxL, and is attenuated by Noxa knockdown loading control. (B) ROS production was measured in SH-EP/Bim cells (Fig. 6A,B). This suggests that ROS accumulation during treated with 200 ng/ml doxy for 0 and 2 hours using MitoTracker Red CM- FOXO3 activation occurs downstream of Bim and BclxL. The H2XROS. (C) Mitochondrial respiration was measured in SH-EP/tetCtr and repression of anti-apoptotic BclxL (Fig. 5A) by FOXO3 might SH-EP/Bim cells after treatment with 200 ng/ml doxy for 0, 2 and 4 hours further impair the ability of the cell to cope with increasing levels (mean values of three independent experiments 6 s.d.; **P,0.01 compared with corresponding controls). of pro-apoptotic BH3-only proteins and ROS levels (Fig. 5B). In this study, we demonstrate in neuronal cells that FOXO3 represses mitochondrial respiration and induces cellular ROS in induced FOXO3 target that is critical for the decline of ROS response to chemotherapy. FOXO3 triggers a biphasic ROS burst levels after the first ROS accumulation. The sestrin family is by perturbation of mitochondrial function through Bim and comprised of three members, SESN1, SESN2 and SESN3, which BclxL, and in parallel, increases ROS-detoxifying capacity have a dual biochemical function: as antioxidant proteins they are through SESN3 (Fig. 8). The first increase in ROS might cause involved in the of peroxiredoxins that scavenge cellular damage that, in combination with prolonged ROS (Budanov et al., 2004) and they also act as inhibitors of neutralization of pro-survival BCL2 proteins, results in a target of rapamycin complex 1 (TORC1) (Chen et al., 2010; secondary stronger ROS burst, which leads to apoptosis. Budanov and Karin, 2008). SESN1 and SESN2 are induced upon Therefore, ROS can be considered as critical mediators of DNA damage and thereby diminish the activity of TORC1. FOXO3-induced apoptosis, where on one hand FOXO3 activates SESN3 is induced by FOXO1 and also represses the activity of detoxification of ROS, but on the other hand it also triggers ROS TORC1 and anabolic metabolism. This results in reduced protein production by induction of pro-apoptotic and repression of pro- and lipid synthesis and thereby saves energy for DNA repair. survival BCL2 proteins. This dual effect of FOXO3 seems Neuronal cells rely on thiol-reducing systems based on critical in the control of stress sensitivity and chemotherapy- thioredoxin and glutathione, which act as reducers of cellular induced cell death in neuronal tumor cells. The combined data peroxides. This detoxification occurs by the transfer of clearly demonstrate that FOXO3-mediated induction of Bim reducing equivalents from NADPH to peroxides involving disrupts mitochondrial respiration, leading to ROS, which are thioredoxin, thioredoxin reductase, and peroxiredoxins (Prx). If critical downstream mediators of FOXO3-induced cell death in peroxiredoxins are overoxidized (Prx-SO2/3H) during oxidative neuronal cells. FOXO3 induces ROS during apoptosis 1201

pLKO-shFOXO3-191617, pLKO-shSESN3-141228 and pLKO-shSESN3- 143446 lentiviral supernatants were used to infect SH-EP and STA-NB15 cells (SH-EP/shFOXO3-17-clone7, SH-EP/shSESN3-28-clone12, SH-EP/shSESN3-46- clone16, NB15/shFOXO3-17 and NB15/shSESN3-46 cells). The empty vector pLKO.1 served as a control (SH-EP/shCtr and NB15/shCtr). pLIB-mycTag- FOXO3-DBD-iresPuro supernatants were used to infect SH-EP and STA-NB15 cells (SH-EP/FOXO3-DBD and NB15/FOXO3-DBD).

Subcellular fractionation, immunoprecipitation and immunoblotting For immunoblot analyses total protein was prepared from 56106 cells resuspended in lysis buffer (50 mM HEPES-NaOH, 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 10% glycerol) with protease and phosphatase inhibitors. Equal amounts of total protein (50 mg/lane) were separated by SDS-PAGE. For subcellular fractionation, 56107 cells were resuspended in MSH buffer (210 mM mannitol, 70 mM sucrose, 20 mM HEPES, 1 mM EDTA, pH 7.4) with protease and phosphatase inhibitors and left on ice for 1 hour. Nuclei and non-lysed cells were removed by centrifugation at 500 g for 5 minutes. To obtain the mitochondrial and cytoplasmic fraction, the supernatant was centrifuged at 25,000 g for 30 minutes. The membrane pellet was resuspended in MSH buffer containing protease inhibitor and 1% CHAPS, lysed on ice for 30 minutes and centrifuged at 25,000 g for 30 minutes. Protein concentration was measured using Bradford Reagent (Bio-Rad Laboratories, Munich, Germany). For immunoprecipitation, 1 mg of rabbit anti-BclxL antibody (Cell Signaling Technology, Boston, MA) or rabbit immunoglobulin, as a negative control, were added to 200 mg mitochondrial lysate and incubated on ice for 2 hours. For pull- down of the immunocomplexes Tachisorb Immunoadsorbent (Calbiochem, Fig. 8. Proposed model for ROS regulation during FOXO3-induced cell Nottingham, UK) was added to the mitochondrial lysate overnight. Tachisorb death in human neuroblastoma cells. FOXO3 activates biphasic ROS immunocomplexes were washed four times in MSH buffer, resuspended in SDS accumulation, which is initiated by induction of pro-apoptotic Bim, repression sample buffer and subjected to SDS-PAGE and blotting. Equal amounts of total of BclxL and consequently displacement of BAX and Bak from BclxL. In protein and cleared supernatants were loaded as controls. After gel separation and parallel, FOXO3 strongly induces the ROS-detoxifying protein SESN3, which blotting, the membranes were blocked, incubated with primary antibodies specific for pBim(Ser69), BclxL, BAX, Bak, CoxIV, JNK, pJNK-T183/T185 (Cell causes transitory ROS decay and prolongs survival during FOXO3 activation. Signaling Technology), Bim, c-Myc (BD-Pharmingen, USA), Catalase (Calbiochem), Noxa (Alexis Biochemicals, San Diego, CA), MnSOD, FOXO3 Materials and Methods (Upstate Biotechnology, Lake Placid, NY), GAPDH (Acris Antibodies, Herford, Cell lines, culture conditions and reagents Germany), SESN3 (Abcam, Cambridge, UK) and a-tubulin (Oncogene Research The neuroblastoma cell lines SH-EP and STA-NB15 (Narath et al., 2005) as well Products, La Jolla, CA), washed and incubated with anti-mouse or anti-rabbit as Phoenix packaging cells for helper-free production of amphotropic retroviruses horseradish-peroxidase-conjugated secondary antibodies. The blots were (Grignani et al., 1998) were cultured in RPMI1640 (Lonza, Basel, Switzerland) developed by enhanced chemiluminescence (GE Healthcare, Vienna, Austria) containing 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin and according to the manufacturer’s instructions and analyzed with a AutoChemi detection system (UVP, Cambridge, UK). Signal intensities were measured using 2 mM L-glutamine (Gibco BRL, Paisley, UK) in 5% CO2. HEK293T cells for production of lentiviruses were cultured in DMEM (Lonza, Basel, Switzerland). LabWorks software (UVP). All cultures were routinely tested for mycoplasma contamination using the Venor GeM-mycoplasma detection kit (Minerva Biolabs, Berlin, Germany). All reagents Live cell fluorescence microscopy Journal of Cell Science were purchased from Sigma (Vienna, Austria) unless indicated otherwise. For live-cell analyses, cells were grown on LabTek Chamber Slides (Nalge Nunc International, Rochester, NY) or on 35 mm optical plastic dishes (Ibidi, Retroviral and lentiviral expression vectors Marinsried, Germany), coated with 0.1 mg/ml collagen. ROS measurements The vectors pBABE-MnSOD-Puro, pLIB-FOXO3(A3)-ER-iresNeo, pLIB-MCS2- were performed by incubating the cells with MitoTracker Red CM-H2XROS iresPuro, pLIB-rtTA-M2-iresTRSID-iresPuro, pQ-tetH1-shBim-SV40Puro, pQ- (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions (final tetH1-shNoxa-SV40Puro, pQ-tetH1-SV40Puro, pLIB-ECFP-FOXO3wt-iresPuro, concentration 500 nM). Images were acquired with an Axiovert200M microscope pLIB-BclxL-iresPuro, pQ-tetCMV-Bim-SV40Neo and pQ-tetCMV-SV40Neo (Zeiss, Vienna, Austria). Fluorescence intensity was quantified using Axiovision have been described previously (Kuznetsov et al., 2008a; Ausserlechner et al., Software (Zeiss) and relative ROS levels were expressed as a percentage of that in 2006; Hagenbuchner et al., 2010; Obexer et al., 2007; Obexer et al., 2009). The untreated controls. lentiviral vectors coding for FOXO3-specific shRNA (pLKO1-shFOXO3-91617), SESN3-specific shRNA (pLKO-shSESN3-141228 and pLKO-shSESN3-143446) Quantitative RT-PCR analysis and the control vector pLKO.1 were obtained from Thermo Scientific (Huntsville, To quantify sestrin-3 (SESN3) mRNA levels, we designed ‘real-time’ RT-PCR AL). pLIB-mycTag-FOXO3-DBD-iresPuro was constructed by inserting the assays, using GAPDH as reference gene. SH-EP/FOXO3 and NB15/FOXO3 cells FOXO3-DBD fragment from pSG5-MycTag-FOXO3-DBD (Dijkers et al., 2002) were cultured in the presence of 100 nM 4OHT for the times indicated. Total RNA into the EcoR1 and Sal1 sites of pLIB-MCS2-iresPuro (Ausserlechner et al., 2006). was prepared from 56106 cells using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. cDNA was synthesized from 1 mgof Production of retroviruses and lentiviruses for infection total RNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (MBI 66105 Phoenix cells were transfected with 2 mg of retroviral vectors and 1 mgofa Fermentas, St Leon-Rot, Germany). Real-time RT-PCR was performed on the plasmid coding for VSV-G protein using Lipofectamine 2000 (Invitrogen, iCycler instrument (Bio-Rad) using sestrin-3 (forward, 59-CCGCCAGTA- Carlsbad, CA) (Ausserlechner et al., 2006; Riml et al., 2004). For production of ACTATCATACATGCG-39 and reverse, 59-GAGGATGTTGACACAACCAT- lentiviruses 6.56105 HEK293T cells were transfected with 1.6 mg pLKO.1 GCTG-39) and GAPDH oligonucleotides (forward, 59-TGTTCGTCATG- vectors expressing FOXO3-specific or SESN3-specific shRNA (Thermo GGTGTGAACC-39 and reverse, 59-GCAGTGATGGCATGGACTGTG-39). All Scientific) and the packaging plasmid pCMV 8.91 (kindly provided by Didier reactions were done in triplicate using a thermal profile of an initial 3 minute Trono, EPFL, Lausanne, Switzerland). After 48 hours, the virus-containing melting step at 95˚C, followed by 40 cycles comprising 95˚C for 20 seconds and supernatants were filtered through 0.22 mm syringe filters (Sartorius, Goettingen, 55˚C for 45 seconds. To verify the presence of only a single amplicon, a melting Germany) and incubated with the target cells for at least 6 hours. SH-EP/ECFP- curve was processed after each run. After normalization to GAPDH expression, FOXO3, SH-EP/FOXO3-shBim-clone1, SH-EP/FOXO3-shNoxa-clone3, NB15/ regulation was calculated between treated and untreated cells. FOXO3-shBim, NB15/FOXO3-shNoxa, SH-EP/tetCtr and SH-EP/Bim cells have already been described (Hagenbuchner et al., 2010; Obexer et al., 2007; Obexer Flow cytometry et al., 2009). pLIB-BclxL-iresPuro supernatants were used to generate SH-EP/ Apoptosis was assessed by staining the cells with propidium iodide (PI) and forward FOXO3-BclxL and NB15/FOXO3-BclxL cells. pBABE-MnSOD-Puro (gift from and sideward scatter analysis using a CytomicsFC-500 Beckman Coulter. 26105 Jakob Troppmair, Medical University Innsbruck, Austria) supernatants were used cells were harvested and resuspended in hypotonic PI solution for 2–4 hours at 4˚C. to infect SH-EP/FOXO3 cells (SH-EP/FOXO3-MnSOD). Stained nuclei in the sub-G1 marker window were considered to represent apoptotic 1202 Journal of Cell Science 125 (5)

cells (Hagenbuchner et al., 2010; Obexer et al., 2007; Obexer et al., 2009). Statistical cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival analysis was performed using GraphPad Prism 4.0 software. through maintenance of mitochondrial integrity. J. Cell Biol. 156, 531-542. Essers, M. A., Weijzen, S., Vries-Smits, A. M., Saarloos, I., de Ruiter, N. D., Bos, J. L. and Burgering, B. M. (2004). FOXO transcription factor activation by Cellular respiration oxidative stress mediated by the small GTPase Ral and JNK. EMBO J. 23, 4802-4812. Oxygen consumption of the cells and mitochondrial function were analyzed by high- Green, D. R. and Kroemer, G. (2004). The pathophysiology of mitochondrial cell resolution respirometry at 30˚C (Kuznetsov et al., 2008b), using a two-channel death. 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