Oncogene (2011) 30, 4557–4566 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE NF-jB suppresses ROS levels in BCR–ABL þ cells to prevent activation of JNK and cell death

SJ Stein and AS Baldwin

Lineberger Comprehensive Center and Department of Biology, University of North Carolina, Chapel Hill, NC, USA

Elevated levels of reactive species (ROS) are results in the creation of the BCR–ABL fusion protein, found in most oncogenically transformed cells and are which is a constitutively active tyrosine kinase (Sawyers, proposed to promote cellular transformation through 1999). As a consequence of increased tyrosine kinase mechanisms such as inhibition of phosphatases. BCR– activity, BCR–ABL phosphorylates substrates, includ- ABL, the oncoprotein associated with the majority of ing Grb2, Crkl and Shc, and activates signaling chronic myeloid leukemias (CMLs), induces accumulation cascades, such as the Ras pathway, PI3K/Akt and of intracellular ROS, causing enhanced signaling down- Stat5, affecting the growth and differentiation of stream of PI3K. Previously we have shown that the myeloid cells (Diaz-Blanco et al., 2007). nuclear factor-kappa B (NF-jB) is Nuclear factor-kappa B (NF-kB) is a transcription activated by BCR–ABL expression and is required for factor, comprised of five family members: p65 (RelA), BCR–ABL-mediated cellular transformation. Inhibition RelB, c-Rel, p50/p105 (NF-kB1) and p52/p100 (NF- of IjB kinase (IKKb) and NF-jB leads to cell death kB2). These proteins share a conserved Rel homology through an unknown mechanism. Here, we analyze the domain, which controls DNA binding, dimerization and potential involvement of NF-jB in moderating BCR– interaction with inhibitory IkB proteins (Courtois and ABL-induced ROS levels to protect from death. The data Gilmore, 2006; Basseres and Baldwin, 2006). NF-kB confirm that BCR–ABL promotes ROS and demonstrate activation typically occurs through one of two distinct that NF-jB prevents excessive levels. Inhibition of NF-jB pathways. In the classical pathway, the p50-p65(RelA) leads to an increase in ROS levels and to cell death, which heterodimer is activated by the IkB kinase (IKK) complex, is at least partially controlled through ROS-induced c-Jun which contains two catalytic subunits, IKKa and IKKb, N-terminal kinase activity. The data demonstrate that one and a regulatory subunit, IKKg. IKK phosphorylates IkBa, function for NF-jB in oncogenesis is the suppression of an inhibitory protein that normally sequesters p50–p65 oncoprotein-induced ROS levels and that inhibition of in the cytoplasm, causing it to become ubiquitinated and NF-jB in some , including CML, will increase ROS subsequently degraded, allowing NF-kB to accumulate levels and promote cell death. in the nucleus. In the alternative pathway, IKKa homo- Oncogene (2011) 30, 4557–4566; doi:10.1038/onc.2011.156; dimers are activated and subsequently phosphorylate published online 30 May 2011 p100. This results in the proteolytic processing of p100 to p52 and allows p52-RelB dimers to translocate to Keywords: IKKb inhibitor; BCR–ABL; ROS; NF-kB; the nucleus (Hayden and Ghosh, 2004). Once in the JNK nucleus, NF-kB is known to regulate the expression of a variety of , including those encoding and receptors, inflammatory mediators and anti-apoptotic proteins (Basseres and Baldwin, 2006). Introduction NF-kB is activated in many solid tumors (Basseres and Baldwin, 2006) and hematological malignancies, Chronic myeloid leukemia (CML) is a malignant clonal including CML (Braun et al., 2006), in which it provides disorder of hematopoietic stem cells that results in proliferative and cell survival mechanisms. NF-kBis increased and deregulated growth of myeloid cells activated by BCR–ABL and is required for cellular (Sawyers, 1999). Approximately 95% of CML cases transformation and tumor formation induced by this arise from the formation of the Philadelphia (Ph) oncoprotein (Hamdane et al., 1997; Reuther et al., , a product of a chromosomal translocation 1998). Inhibition of IKK in BCR–ABL-expressing cells that brings together the c-abl on chromosome 9 induces death (Cilloni et al., 2006; Duncan et al., 2008). and the bcr gene on chromosome 22. This translocation Interestingly, Imatinib- and/or Dasatinib-resistant cells were shown to be susceptible to IKKb inhibition Correspondence: Dr AS Baldwin, Lineberger Comprehensive Cancer (Duncan et al., 2008), suggesting a novel therapeutic Center and Department of Biology, University of North Carolina option for CML. However, the mechanism whereby School of Medicine, 22-000 LCCC, CB#7295, Chapel Hill, NC 27599, IKKb inhibition induces death of BCR–ABL-expressing USA. E-mail: [email protected] cells has not been determined. Received 1 November 2010; revised and accepted 3 April 2011; published c-Jun N-terminal kinase (JNK), also known as online 30 May 2011 stress-activated protein kinase, is a member of the NF-jB suppresses ROS levels in BCR–ABL þ cells SJ Stein and AS Baldwin 4558 mitogen-activated protein kinase family and is involved Dasatinib (Duncan et al., 2008; Cilloni et al., 2006). in the regulation of c-jun, a component of the AP-1 That data showed the importance of IKKb in family of transcription factors (Leppa and Bohmann, BCR–ABL-induced oncogenesis. However, a mechan- 1999). JNK is predominately activated by cellular stress ism mediating IKK inhibitor-induced cell death mechanisms, including increased levels of reactive and involvement of NF-kB in cell survival was oxygen species (ROS), but can also be activated by not shown. As analyzed before, cell viability was other stimuli including cytokines and oncogenic trans- measured to determine the effect of IKKb inhibition formation. JNK is activated by MAP kinase kinases using Compound A (a well-validated IKKb inhibitor, through the phosphorylation of threonine 183 and Ziegelbauer et al., 2005) in parental 32D cells and in tyrosine 185. JNK then phosphorylates c-Jun at serines 32D cells stably expressing BCR–ABL p185 (32D/p185). 63 and 73 causing an increase in c-Jun transcriptional Compound A treatment resulted in decreased cell activity (Gupta et al., 1996). c-Jun activity is implicated viability similar to treatment with Imatinib, whereas in cell transformation, proliferation and death down- Compound C, an inactive analog of Compound A, stream of JNK (Leppa and Bohmann, 1999; Vogt, did not affect the viability of 32D/p185 cells (Figure 1a 2001). Interestingly, both c-jun and JNK are required and see Duncan et al., 2008). The decrease in cell for transformation of hematopoietic cells by BCR–ABL viability with Compound A treatment corresponds with (Raitano et al., 1995) as well as their survival after cleavage of caspase 3, a marker of apoptosis (Figure 1b). transformation (Hess et al., 2002). However, under Similar results were seen in parental BaF3 pro-B stimuli that induce cell stress, JNK activation can lead cells and BaF3 cells-expressing BCR–ABL (Supplemen- to death (Shen and Liu, 2006; Dhanasekaran and tary Figure S1). Co-incubation with ZVAD-FMK, Reddy, 2008). JNK becomes activated by stimuli in a an inhibitor of caspase activation, potently blocks constitutive manner through increased intracellular Compound A-induced cell death. These results show ROS, and activates apoptotic and necrotic death path- that IKKb activity is required to block apoptosis in ways (Tang et al., 2002; Pham et al., 2004; Ventura cells expressing BCR–ABL (Figure 1b and Duncan et al., 2004; Kamata et al., 2005). et al., 2008). It has been demonstrated that oncogenic transforma- tion results in increased levels of intracellular ROS, which NF-kB activity is required for the survival are used as secondary signaling molecules to increase of BCR–ABL-expressing cells proliferation and to promote the oncogenic potential of Although IKKb is known to activate NF-kB through the transformed cells (Benhar et al., 2002; Pelicano et al., phosphorylation-mediated ubiquitination and degradation 2004). For example, oncogenic Ras leads to increased levels of IkBa, it also has other targets (Hu et al., 2004; Lee et al., of ROS, which are important in oncogenic transformation 2007). Therefore, to determine whether NF-kB is necessary and proliferation (Irani et al., 1997). Previous reports have for the survival of BCR–ABL-expressing cells downstream shown that hematopoietic cell lines transformed with of IKKb, and to rule out off-target effects of Compound BCR–ABL have increased levels of intracellular ROS A, NF-kB activity was blocked by expressing IkBa super- (Sattler et al., 2000; Kim et al., 2005; Naughton et al., repressor (IkBa-SR), a form of IkBa containing serine to 2009). ROS promotes PI3K-induced signaling downstream alanine mutations at residues 32 and 36 that prevent its of BCR–ABL by inhibiting phosphatases, which normally phosphorylation and degradation, thereby sequestering limit signal transduction cascades (Naughton et al., 2009), NF-kB in the cytoplasm of the cell. Expression of IkBa- thereby increasing tumorigenicity. SR led to apoptosis in BCR–ABL-expressing 32D cells In this study, we have explored the potential involve- over time as measured by Annexin V/propidium iodide ment of NF-kB in moderating intracellular ROS levels staining (Figure 2a) and expression of cleaved caspase 3 downstream of BCR–ABL. The results indicate that (Figure 2b), whereas the viability of cells transduced NF-kB activity functions to suppress BCR–ABL-induced with empty vector was not affected. Taken together, ROS levels. In addition, inhibition of IKK or NF-kB these results show a requirement for NF-kB activity leads to enhanced ROS levels and elevated JNK activity downstream of IKKb in hematopoietic cells expressing to promote cell death. The experiments reveal a key pro- BCR–ABL to prevent apoptosis. oncogenic mechanism and demonstrate a mechanism whereby inhibition of NF-kB activity promotes cyto- toxicity of certain cancer cells. IKKb inhibition in BCR–ABL-expressing cells results in the accumulation of intracellular Although the inhibition of both IKKb and NF-kBin Results BCR–ABL-expressing cells results in apoptosis, the mechanism that precedes cell death remains unclear. Inhibition of IKKb results in apoptosis Cells that have undergone oncogenic transformation, of BCR–ABL-expressing cells including those overexpressing Ras, c-myc and BCR– Recently, we and others have shown that IKKb activity ABL, have increased levels of intracellular ROS (Irani is required for survival of BCR–ABL-expressing mye- et al., 1997; Sattler et al., 2000; Vafa et al., 2002). loid cells, including cells with mutations resistant to the Transformed cells utilize increased ROS as secondary commonly used BCR–ABL inhibitors Imatinib and signaling molecules to enhance proliferation and tumor

Oncogene NF-jB suppresses ROS levels in BCR–ABL þ cells SJ Stein and AS Baldwin 4559

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AV Figure 1 Inhibition of IKKb causes apoptosis in BCR–ABL-expressing myeloid cells. (a) 32D cells expressing BCR–ABL (32D/p185) were incubated with 1 mM Imatinib, IKK inhibitor (Compound A) or an inactive analog of Compound A (Compound C) for 20 h. Cell viability was measured by MTS reduction. (b) 32D/p185 cells were co-incubated with Z-VAD-FMK (100 mM) and DMSO (vehicle control) or 1 mM Compound A for 20 h. Cells were stained with propidium iodide and Annexin V and cell death was measured by fluorescence-activated cell sorting analysis or (c) lysed and analyzed by immunoblot.

vector SR vector SR

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AV Figure 2 NF-kB activity is required for survival of BCR–ABL-expressing cells. (a) 32D/p185 cells were transduced with retrovirus expressing vector control or IkBa superrepressor (IkBa-SR). Cells were collected at 36 h after retroviral transduction and stained with propidium iodide and Annexin V. Cell death was analyzed by fluorescence-activated cell sorting, or (b) analyzed by immunoblot. development. However, because transformed cells har- et al., 2004). As BCR–ABL expression is known to bor higher levels of ROS, a further increase in free enhance reactive oxygen species production in hemato- radicals can result in apoptosis or (Pelicano poietic cells (Sattler et al., 2000; Kim et al., 2005;

Oncogene NF-jB suppresses ROS levels in BCR–ABL þ cells SJ Stein and AS Baldwin 4560

Figure 3 Inhibition of NF-kB causes an increase in intracellular ROS in cells expressing BCR–ABL. (a) 32D/p185 cell were incubated with DMSO (vehicle control) or 1 mM Imatinib or Compound A for 16 h without or (b) with antioxidants. Cells were then incubated with DCFDA and fluorescence was measured by fluorescence-activated cell sorting.

6 hr 12 hr 16 hr 24 hr vector SR DMSO CpA DMSO CpA DMSO CpA DMSO CpA vector SR p-JNK (T183/Y185) p-JNK (T183/Y185) p-JNK (T183/Y185) p-c-jun (S73) clvd. casp. 3 p-c-jun (S73) clvd. casp. 3 IκBα clvd. casp. 3 IκBα JNK JNK JNK Actin c-jun c-jun casp. 3 casp. 3 β tubulin Actin

Figure 4 IKKb inhibition downstream of BCR–ABL induces JNK activation and apoptosis. (a) 32D/p185 cells were treated with DMSO or 1 mM Compound A over a time course. Cells were lysed and subjected to immunoblotting. (b) 32D/p185 or (c) parental 32D cells were transduced with retrovirus expressing vector control or IkBa superrepressor (IkBa-SR) and collected at 36 h after retroviral transduction, lysed and subjected to immunoblotting.

Naughton et al., 2009) and NF-kB can regulate to the accumulation of ROS in these cells. The antioxidant (Tang et al., 2002; Pham accumulation of ROS upon treatment with Compound et al., 2004; Kamata et al., 2005), we asked whether A is reversed through the addition of antioxidants n- IKKb inhibition with Compound A results in altered acetyl-cysteine (NAC) or butylated hydroxyanisole ROS levels leading to cell death. Relative ROS levels (BHA) (Figure 3b). These data indicate that IKKb were measured in 32D/p185 cells treated with Imatinib inhibition leads to significantly enhanced levels of ROS, or Compound A over time. Treatment with the BCR– over those induced by BCR–ABL. ABL inhibitor Imatinib decreased intracellular ROS levels (Figure 3a) as previously reported (Sattler et al., 2000), whereas IKKb inhibition using Compound A NF-kB inhibits the activation of JNK downstream caused an increase in intracellular ROS as measured by of BCR–ABL to promote cell survival DCFDA staining. Cells treated for 12 h (data not At high levels, ROS have been shown to activate AP-1, shown) or 16 h showed an accumulation of ROS resulting in cell death (Shen and Liu, 2006). Interest- (Figure 3), whereas cells treated for 1 h did not (data ingly, NF-kB is important for the regulation of JNK, an not shown), suggesting that an indirect mechanism leads upstream effector of AP-1, to block death under cell

Oncogene NF-jB suppresses ROS levels in BCR–ABL þ cells SJ Stein and AS Baldwin 4561

Figure 5 NF-kB regulates antioxidant gene expression in BCR–ABL-expressing cells. (a) 32D/p185 cells were treated for 12 h with DMSO or 1 mM Compound A. RNA was purified using Trizol and then subjected to reverse transcription before analysis using quantitative real-time PCR. (b) 32D/p185 cells were transduced with retrovirus expressing empty vector or IkBa-SR. Gene expression was analyzed by quantitative real-time PCR as described. (c) Expression levels of IkBa of cells used in panel B.

stress conditions (Tang et al., 2002; Pham et al., 2004; IKKb inhibition leads to downregulation of antioxidant Kamata et al., 2005). Given the correlation between gene transcription increased intracellular ROS and apoptosis in BCR– Our results show that NF-kB activity is important for ABL-expressing cells after Compound A treatment, we the regulation of intracellular ROS and JNK activity asked whether NF-kB activation is important for the downstream of BCR–ABL to prevent cells from under- regulation of intracellular ROS and inhibition of JNK going apoptosis. NF-kB is known to regulate the downstream of BCR–ABL. A time course in which 32D/ expression of genes encoding proteins with antioxidant p185 cells were treated with Compound A shows that properties (Pham et al., 2004; Kamata et al., 2005). both the phosphorylation of JNK, its downstream Owing to the increase in intracellular ROS upon target c-jun, and caspase-3 cleavage occur 6 h after inhibition of IKKb, we asked if NF-kB transcriptionally treatment (Figure 4a). 32D/p185 cells were transduced regulates genes known to clear excess ROS from the cell. with empty vector or IkBa-SR to examine the effect of BCR–ABL-expressing cells were treated with vehicle or NF-kB inhibition on JNK activation and apoptosis Compound A and quantitative real-time PCR was used downstream of BCR–ABL. Cells collected at 36 h post- to screen NF-kB target genes known to have antioxidant transduction showed increased phosphorylation of properties. 32D/p185 cells treated with Compound A for JNK, c-jun and the cleavage of caspase 3 (Figure 4b). 12 h showed decreased levels of both Sod2 and Fth1 Parental 32D cells expressing IkBa-SR were not affected mRNAs (Figure 5a), corresponding with the phosphor- to the same extent as 32D/p185 cells, although some ylation of JNK and apoptosis (Figure 4a). This result apoptosis is apparent (Figure 4c). This low level of cell indicates that blocking IKKb activity leads to decreased death can be attributed to moderate activation of NF- production of two known ROS scavengers, possibly kB in these cells because of their dependence on IL-3 for resulting in accumulation of intracellular ROS and survival (Reuther et al., 1998). Although IL-3 is also apoptosis. To rule out potential off-target effects of known to activate JNK (Yu et al., 2004), expression of Compound A, IkBa-SR was overexpressed to block IkBa-SR did not affect JNK phosphorylation in these NF-kB activity in 32D/p185 cells. Similar to the results cells. Together, these data show that NF-kB actively obtained using Compound A treatment, cells expressing regulates the level of intracellular ROS and also inhibits IkBa-SR (Figure 5c) also showed decreased mRNA the activation of JNK downstream of BCR–ABL to levels of Sod2 and Fth1 (Figure 5b), correlating with prevent cells from undergoing apoptosis. apoptosis. Overexpression of Sod2 and Fth1 did not

Oncogene NF-jB suppresses ROS levels in BCR–ABL þ cells SJ Stein and AS Baldwin 4562

SP600125: 0 μM 10 μM25μM DMSO CpA DMSO CpA DMSO CpA

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Figure 6 JNK activation in the absence of NF-kB activity leads to apoptosis in BCR–ABL-expressing cells. (a) 32D/p185 cells were pretreated with vehicle or indicated concentrations of SP600125 for 1 h. Compound A (1 mM) or DMSO was then added and cells were incubated for an additional 24 h. Cells were then collected, lysed and subjected to immunoblot. (b) 32D/p185 cells were incubated with vehicle or 25 mM SP600125 for 1 h before the addition of DMSO of 1 mM Compound A for an additional 20 h. Cells were stained using Annexin V and propidium iodide. Cell death was measured by fluorescence-activated cell sorting. (c) 32D/p185 cells were transduced with IkBa-SR. 20 h post-transduction, cells were treated with SP600125 for an additional 8 h. Cells were then lysed and analyzed by immunoblot.

rescue the cell death response induced by IKKb for cell death in BCR–ABL-expressing cells when NF-kB inhibition (data not shown), suggesting that multiple is inhibited. These data further suggest an important role mechanisms controlled by IKK and NF-kB contribute for JNK regulation and evasion of apoptosis through to the regulation of ROS levels in oncogenically NF-kB downstream of BCR–ABL. transformed cells.

Inhibition of ROS prevents death due to IKKb JNK inhibition rescues BCR–ABL þ cells from apoptosis inhibition in BCR–ABL þ cells after Compound A treatment The increase in intracellular ROS in transformed cells Our results show that NF-kB activity regulates intra- enhances proliferation and tumorigenicity. However, these cellular ROS levels and JNK activation in BCR–ABL- cells are also sensitive to further increases in intracellular expressing cells. To determine the importance of JNK ROS, which may lead to apoptosis (Pelicano et al., activity in the death of BCR–ABL-expressing cells after 2004). Our data show that inhibition of NF-kB leads inhibition of NF-kB, we blocked JNK using a specific to a further increase in intracellular ROS, activation inhibitor, SP600125, and treated 32D/p185 cells with of JNK and apoptosis downstream of BCR–ABL. To Compound A. Cells that were treated with SP600125 better understand the role of NF-kB in the regulation and Compound A showed decreased apoptosis as indi- of intracellular ROS in cells expressing BCR–ABL, we cated by caspase 3 cleavage (Figure 6a) and fluorescence- inhibited ROS and measured cell death after Compound activated cell sorting analysis (Figure 6b). However, cells A treatment. Interestingly, 32D/p185 cells incubated treated with high concentrations of SP600125 (X25 mM) with NAC or BHA in conjunction with Compound A underwent apoptosis without IKKb inhibition, indicating treatment showed a pronounced decrease in phosphory- that BCR–ABL-expressing cells also require low levels lated JNK and were resistant to apoptosis (Figures 7a of JNK activity for survival as previously shown and b). Similar results were obtained in Ba/F3 cells (Raitano et al., 1995; Hess et al., 2002). Similar results expressing BCR–ABL (Supplementary Figure S2). were obtained from 32D/p185 cells that were treated Cells were also co-incubated with bovine catalase, an with SP600125 upon expression of IkBa-SR (Figure 6c). antioxidant , and Compound A, resulting in These data show that increased JNK activity is required decreased JNK phosphorylation and apoptosis

Oncogene NF-jB suppresses ROS levels in BCR–ABL þ cells SJ Stein and AS Baldwin 4563 NAC BHA DMSO CpA DMSO CpA CpA DMSO CpA DMSO none p-JNK (T183/Y185)

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Catalase: 0 100 250 NAC vector SR vector SR CpA DMSO CpA DMSO CpA DMSO p-JNK (T183/Y185) p-JNK (T183/Y185) IκBα clvd. casp. 3 clvd. casp. 3 JNK JNK casp. 3 casp. 3 Actin Actin

Figure 7 Antioxidants rescue BCR–ABL-expressing cells from death when NF-kB is inhibited. 32D/p185 cells were pretreated with vehicle, 20 mM NAC or 50 mM butylated hydroxyanisole for 1 h before the addition of DMSO or 1 mM Compound A. Cells were incubated for an additional 20 h and (a) lysed and subjected to immunoblot, or (b) stained with Annexin V and propidium iodide and analyzed using flow cytometry. (c) 32D/p185 cells were pretreated with bovine catalase for 1 h before the addition of DMSO or 1 mM Compound A. Cells were incubated for an additional 20 h and then collected for immunoblot. (d) 32D/p185 cells were transduced with retrovirus expressing empty vector or IkBa-SR. 24 h after transduction, cells were replated in media with or without 20 mM NAC for an additional 12 h. Cells were collected, lysed and used for immunoblotting. (e). Model showing the role of NF-kB in the regulation of intracellular ROS in BCR–ABL-expressing cells.

(Figure 7c). Lastly, 32D/p185 cells were incubated with and Nathan, 1991), leading to the hypothesis that cancer NAC upon expression of IkBa-SR. JNK activation and cells are subject to persistent , possibly apoptosis induced by the overexpression of IkBa-SR explaining characteristics of cancer including genomic were also inhibited by NAC treatment (Figure 7d). instability and increased proliferation (Toyokuni et al., These results show that NF-kB activity is required to 1995, Trachootham et al., 2009). Indeed, several reports regulate increased intracellular ROS following transfor- have shown an increase in reactive oxygen species in mation with BCR–ABL. Upon inhibition of NF-kB, the primary human tumors, including brain (Iida et al., accumulation of ROS in the cell leads to the activation of 2001), colorectal carcinoma (Toyokuni et al., 1995), and JNK and apoptosis (Figure 7e). ovarian cancer (Senthil et al., 2004). In addition, reports showed that oncogenic transformation by Ras, c-myc and BCR–ABL lead to increased ROS, which is important for increased proliferation and tumorigenic Discussion potential (Irani et al., 1997; Felsher and Bishop, 1999; Vafa et al., 2002; Sattler et al., 2000; Kim et al., 2005; Increased ROS has been documented in several cell Naughton et al., 2009). types after oncogenic transformation and in various Relative to oncogenic Ras expression, increased ROS cancers (Benhar et al., 2002; Pelicano et al., 2004). It levels were shown to be required for cellular trans- was first discovered that human tumor cells produce formation (Irani et al., 1997). In this regard, ROS increased amounts of (Szatrowski generated from the Qo site of mitochondrial complex

Oncogene NF-jB suppresses ROS levels in BCR–ABL þ cells SJ Stein and AS Baldwin 4564 III is required for anchorage-independent growth of and apoptosis (Figure 4b). These data correlate with Ras-transformed cells (Weinberg et al., 2010). Over- previous reports in which NF-kB has an important role expression of Nox1, a generator, in NIH3T3 in JNK inhibition when ROS levels increase (Sakon cells results in elevated production of ROS and a et al., 2003; Pham et al., 2004; Kamata et al., 2005). transformed phenotype with increased proliferation Treatment with Compound A or expression of IkBa- (Suh et al., 1999). Interestingly, Nox1 knockdown SR also results in decreased expression of two NF-kB blocks Ras-transformed phenotypes including anchorage target genes with antioxidant properties, Fth1 and Sod2 independent growth in vitro and in vivo (Mitsushita et al., (Figure 5). These genes have been documented in 2004). Relative to our study, ROS levels are increased response to tumor necrosis factor-a stimulation in which downstream of BCR–ABL which leads to increased tumor necrosis factor-a-induced ROS was scavenged, PI3K/Akt-dependent signaling through inhibition of the protecting cells from tumor necrosis factor-a-induced phosphatase PP1a (Naughton et al., 2009). death in the absence of NF-kB (Pham et al., 2004; Cells transformed with BCR–ABL have high levels of Kamata et al., 2005). Although inhibition of ROS (Sattler et al., 2000; Kim et al., 2005; Naughton NF-kB results in decreased antioxidant gene expression et al., 2009) (and see Figure 3a), thus enhancing the (Fth1 and Sod2), our preliminary data indicates that sensitivity of these cells to a further increase in ROS. overexpression of either FTH1 or SOD2 in BCR–ABL- Treatment with agents that cause an increase in ROS in expressing cells is not sufficient to inhibit apoptosis in BCR–ABL-expressing cells leads to death (Zhou et al., the absence of NF-kB activity. This is not surprising, as 2003; Chandra et al., 2006; Trachootham et al., 2006, many cellular processes control the levels of ROS, Mao et al., 2008; Zhang et al., 2008). One such agent, indicating that other NF-kB-dependent genes and phenethyl isothiocyanate results in increased ROS buffering systems are likely involved in this process. and subsequent apoptosis in cells expressing both Our data also show that JNK activity is involved in wild-type (Trachootham et al., 2006) and Imatinib- the initiation of apoptosis in the absence of NF-kB. and Dasatinib-resistant (Zhang et al., 2008) forms of Blocking JNK activity with a chemical inhibitor, BCR–ABL. However, the mechanism by which these SP600125, results in a decrease in death upon Com- compounds cause increased ROS and cell death is pound A treatment in BCR–ABL-expressing cells. largely unknown. (Figure 6). However, cells expressing BCR–ABL appear Data described above indicate that the maintenance to require JNK activity, as the inhibitor alone results in of moderate levels of ROS are necessary for increased induction of apoptosis in 32D/p185 cells. Importantly, proliferative capacity and tumorigenic potential while JNK activation by ROS is required for the initiation of avoiding death in response to excessive accumulation apoptosis in the absence of NF-kB activity (Figure 7). of free radicals (Benhar et al., 2002; Schimmel and However, inhibition of ROS with antioxidants offers Bauer, 2002; Pelicano et al., 2004). Owing to excessive more complete protection from Compound A-induced strain on ROS clearing mechanisms that maintain a apoptosis than inhibition of JNK with SP600125. This moderate balance of ROS, a further increase in ROS in could simply be because of the efficiency of inhibition by transformed cells may result in cancer cell death (Benhar these compounds, or the differences in survival could et al., 2002; Pelicano et al., 2004), offering a novel indicate a more involved role for increased ROS in approach to target cancer cells. apoptosis after inhibition of NF-kB. It is probable that Potential therapeutic targets that may increase ROS, ROS activate JNK as well as other proteins in the cell to specifically in cancer cells, include transcription factors initiate apoptosis in response to unfavorable conditions, that control the expression of both antiapoptotic and that inhibiting JNK only partially blocks the effect and antioxidant genes. One such transcription factor, of increased ROS on cell survival. NF-kB, has been shown to regulate the transcription of These data show that NF-kB is required to maintain genes with antioxidant properties, such as ferritin heavy moderate levels of ROS and inhibit JNK activation chain (Pham et al., 2004) and downstream of BCR–ABL-induced ROS to inhibit the (Kamata et al., 2005). NF-kB also inhibits JNK induction of apoptosis in a model of CML. As increased activation downstream of ROS through transcription ROS is common among transformed cells, it is likely of genes such as Gadd45 (De Smaele et al., 2001) and that NF-kB has an essential role in the regulation of XIAP (Tang et al., 2001) and through the inhibition of ROS to prevent death, illustrating the potential use for mitogen-activated protein kinase (Kamata et al., 2005) IKKb inhibitors as a therapeutic in CML and possibly and tyrosine phosphatases (Sattler et al., 2000). other cancers. Our results show an important role for NF-kB activity in the maintenance of intracellular ROS and the inhibition of JNK activity downstream of BCR– Materials and methods ABL to prevent cell death after oncogenic transforma- tion. Inhibition of IKKb using a chemical inhibitor, Cell lines 32D and Ba/F3 hematopoietic murine cells were maintain in Compound A, results in apoptosis (Figure 1), along with RPMI 1640 medium (Gibco, Grand Island, NY, USA) supple- the accumulation of intracellular ROS (Figure 3) and mented with 10% fetal bovine serum and 10% Wehi- the activation of JNK in BCR–ABL-expressing cells conditioned media as a source of IL-3. 32D and Ba/F3 cells (Figure 4). Similarly, expression of IkBa-SR, which stably expressing p185 or p210 BCR–ABL, respectively, were blocks NF-kB activity, induces JNK phosphorylation maintained in RPMI 1640 supplemented with 10% fetal

Oncogene NF-jB suppresses ROS levels in BCR–ABL þ cells SJ Stein and AS Baldwin 4565 bovine serum. 293Ts were maintained in Dulbecco’s modified Indianapolis, IN, USA). Cells were incubated on ice for 15 min Eagle’s medium supplemented with 10% fetal bovine serum. and the lysates were clarified by centrifugation. Equal amounts of lysates (30–50 mg) were subjected to SDS–PAGE, Chemicals transferred onto a nitrocellulose membrane, blocked for 1 h 20,70-Dichlorodihydrofluorescein diacetate (DCFDA; at room temperature in Tris–buffered saline with 0.05% Calbiochem, Gibbstown, NJ, USA) was dissolved in dimethyl Tween-20 and 5% non-fat milk and incubated with sulfoxide. Catalase and n-acetyl-cysteine (Sigma, St. Louis, the indicated antibodies overnight. Blots were incubated MO, USA) were dissolved in culture media. The pH of NAC with the appropriate secondary antibody for 45 min at was then adjusted to 7.2 and the stock was subsequently room temperature and developed using ECL detection reagent passed through a 0.2-mm filter. Butylated hydroxyanisole (GE, Buckinghamshire, UK). (Sigma) was dissolved in ethanol. Compound A, SP600125 (Sigma) and Z-VAD-FMK (Sigma) were dissolved in dimethyl Quantitative Real-time PCR sulfoxide. All stocks were diluted to working dilutions in Total RNA was isolated using TRIzol reagent (Invitrogen, culture media. Carlsbad, CA, USA), digested with DNase I (Promega, Madison, WI, USA), and used for reverse transcription (SuperScript II Detection of ROS kit, Invitrogen). All Taqman primers were obtained from Cells were collected, washed twice with phosphate-buffered Applied Biosystems (Foster City, CA, USA). Expression levels saline and then incubated with DCFDA at a final concentra- of GusB were used to normalize the amount of the investigated tion of 10 mM for 15 min at 37 1C in the dark. Cells were then transcripts. washed once with phosphate-buffered saline and analyzed immediately by flow cytometry. Viral production and transduction Virus was produced by transient transfection of 293T cells with Cell death staining pCL-10A1 (Imgenex, San Diego, CA, USA) and a retroviral Cells were collected and washed twice with cold phosphate- vector using Fugene at a 1:1 ratio. Viral supernatant was buffered saline. Cells (5 Â 105) were resuspended in 100 ml collected 24 and 48 h post-transfection and concentrated using Annexin-binding buffer (BD Pharmingen, San Diego, CA, centrifugal filter units (Amicon Ultra, Millipore, Billerica, 6 USA) and stained with Annexin V (BD Pharmingen; 1:20) MA, USA). Target cells were resuspended at 0.5 Â 10 cells/ml and Propidium Iodide (BD Pharmingen; 1:20) at room in RPMI with viral supernatant in six-well plates and spun at temperature in the dark for 15 min. A total volume of 400 ml 2500 r.p.m. for 1 h at room temperature. Cells were incubated binding buffer was subsequently added and the cells were with viral supernatant for an additional 3 h at 37 1C and then analyzed immediately by flow cytometry. plated in RPMI for an additional 24–48 h before collecting for experiments. Antibodies Phospho-JNK (T183/Y185), JNK, Phospho-c-jun (S73), c-jun, and cleaved caspase 3 (Asp175), caspase 3 and IkBa Conflict of interest were obtained from Cell Signaling Technologies (Danvers, MA, USA). b-Tubulin was obtained from Santa Cruz Biotech- The authors declare no conflicts of interest. nology (Santa Cruz, CA, USA). b-actin was obtained from Calbiochem. Acknowledgements Western blotting Cells were collected, washed twice with cold phosphate- We thank Justina Chen for technical support and members of buffered saline and resuspended in lysis buffer (50 mM Tris– the Baldwin lab for advice and discussion. We acknowledge HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxy- grant support from the Leukemia and Lymphoma Society, cholate, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4) supple- from the NIH (CA73756 and CA75080), and from the Samuel mented with protease and phosphatase inhibitors (Roche, Waxman Cancer Research Foundation.

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