Selective inhibition of STAT3 induces apoptosis and G1 cell ... - Nature

Oncogene (2004) 23, 5426–5434

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Selective inhibition of STAT3 induces apoptosis and G1 cell cycle arrest in ALK-positive anaplastic large cell lymphoma Hesham M Amin*,1, Timothy J McDonnell2, Yupo Ma1, Quan Lin1, Yasushi Fujio3, Keita Kunisada3, Vasiliki Leventaki1, Pamela Das1, George Z Rassidakis1, Cathy Cutler1, L. Jeffrey Medeiros1 and Raymond Lai*,1 1

Department of Hematopathology, Box 72, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA; 2Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; 3Department of Molecular Medicine, Graduate School of Medicine, Osaka, Japan

Nucleophosmin-anaplastic lymphoma kinase (NPMALK) is an aberrant fusion gene product expressed in a subset of cases of anaplastic large cell lymphoma (ALCL). It has been shown that NPM-ALK binds to and activates signal transducer and activator of transcription 3 (STAT3) in vitro, and that STAT3 is constitutively active in ALK þ ALCL cell lines and tumors. In view of the oncogenic potential of STAT3, we further examined its biological significance in ALCL using two ALK þ ALCL cell lines (Karpas 299 and SU-DHL-1) and an adenoviral vector that carries dominant-negative STAT3 (AdSTAT3DN). Infection by AdSTAT3DN led to the expression of STAT3DN in both ALK þ ALCL cell lines at a similar efficiency. Subcellular fractionation studies showed that a significant proportion of the expressed STAT3DN protein translocated to the nucleus, despite the fact that STAT3DN has a mutation at residue 705tyrosine-phenylalanine, a site that is believed to be crucial for STAT3 activation and nuclear translocation. Introduction of STAT3DN induced apoptosis and G1 cell cycle arrest. Western blot studies showed that expression of STAT3DN resulted in caspase-3 cleavage, downregulation of Bcl-2, Bcl-xL, cyclin D3, survivin, Mcl-1, c-Myc and suppressor of cytokine signaling 3. These results support the concept that STAT3 activation is pathogenetically important in ALCL cells by deregulating the expression of multiple target proteins that are involved in the control of apoptosis and cell cycle progression. Oncogene (2004) 23, 5426–5434. doi:10.1038/sj.onc.1207703 Published online 7 June 2004 Keywords: STAT3; anaplastic large cell lymphoma; NPM-ALK; adenoviral vectors; apoptosis; cell cycle

*Correspondence: HM Amin; E-mail: [email protected] and R Lai is now with Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta and Cross Cancer Institute, 4B1 Walter MacKenzie Health Science Center, 8440112 Street, Edmonton, Alberta, Canada T6G 2B7; E-mail: [email protected] Received 19 June 2003; revised 2 March 2004; accepted 3 March 2004; published online 7 June 2004

Introduction Anaplastic large cell lymphoma (ALCL), first described in 1985 (Stein et al., 1985), is recognized as a distinct type of non-Hodgkin’s lymphoma in various lymphoma classification schemes, including the one most recently published by the World Health Organization (Delsol et al., 2001). These tumors are characterized by CD30 expression, sinusoidal infiltrative pattern, and T/nullcell immunophenotype. A substantial subset of these tumors have the chromosomal translocation, t(2;5)(p23;q35), that juxtaposes the nucleophosmin (NPM) gene at 5q35 with the anaplastic lymphoma kinase (ALK) gene at 2p23, leading to the expression of the aberrant fusion protein NPM-ALK (Morris et al., 1994). ALK expression in ALCL is clinically important, since ALK þ tumors are associated with younger patient age and a better clinical outcome as compared with ALK tumors (Falini et al., 1999; Gascoyne et al., 1999). There is evidence that NPM-ALK contributes to the pathogenesis of ALCL, since deregulation of ALK expression transforms rodent cells and induces IL-3 independence in BaF3 cells (Bai et al., 1998). The mechanism mediating the oncogenic potential of NPM-ALK is not completely understood. ALK embodies a tyrosine kinase domain (Iwahara et al., 1997; Morris et al., 1997) and NPM is a nonribosomal RNAbinding protein that functions in bidirectional shuttling of proteins between the nucleus and cytoplasm (Borer et al., 1989). The pathogenetic role of NPM in ALCL is believed to lie in its ability to oligomerize, thereby bringing ALK together, that leads to autophosphorylation and activation of the ALK tyrosine kinase activity (Bischof et al., 1997). There is evidence that the NPMALK fusion protein mediates oncogenesis by inducing tyrosine phosphorylation of various protein targets, such as Grb2, Shc, IRS-1, PLCg and PI3K (Bai et al., 1998; Duyster et al., 2001; Slupianek et al., 2001), although the biologic significance of these findings needs to be verified. More recently, it has been shown that NPM-ALK binds to, phosphorylates, and activates STAT3 (signal transducer and activator of transcription 3) in vitro (Zamo et al., 2002). In the same study, the authors found that STAT3 phosphorylation/activation

Selective inhibition of STAT3 in ALCL HM Amin et al

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is closely linked to ALK expression in non-Hodgkin’s lymphoma. STATs are members of a ubiquitously expressed family of transcription factors activated in response to growth factors and cytokines (Darnell, 1997; Bowman et al., 2002). STAT3 has been shown to be an oncogene (Bromberg et al., 1999), and many types of human cancer express constitutively active STAT3 (Chai et al., 1997; Garcia et al., 1997; Gouilleux-Gruart et al., 1996; Grandis et al., 1998). A number of downstream targets of STAT3 have been identified in various cell types, many of which are associated with the regulation of apoptosis and cell proliferation. Zamo et al. (2002) found that transfection of NPM-ALK in myeloma cell lines led to STAT3 activation and upregulation of the anti-apoptotic protein Bcl-xL. These findings correlated with increased resistance to apoptosis in these cells (Zamo et al., 2002). Other potential downstream targets of STAT3 have been identified in a variety of cell types, including Mcl-1, cyclin D3, c-Myc and p21waf-1 (Darnell, 1997). Considering the ability of NPM-ALK to activate STAT3 and the biologic importance of STAT3, it is possible that STAT3 activation is important in mediating the oncogenic effects of NPM-ALK. The purpose of this study is to examine the pathogenetic importance of STAT3 activation in two well-characterized ALK þ ALCL cell lines, Karpas 299 and SU-DHL-1, by blocking STAT3 signaling using an adenoviral vector carrying dominant-negative STAT3 (AdSTAT3DN). We analysed the mechanisms underlying the biological responses to STAT3DN in these cell lines by evaluating potential downstream targets of STAT3.

Results Expression of STAT3DN in ALCL cell lines Western blot studies showed that adenoviral gene transfer of STAT3DN resulted in high levels of STAT3DN expression in both Karpas 299 and SUDHL-1. As illustrated in the upper panel of Figure 1, lysates prepared from Karpas 299 cells at 24 h after adenoviral infection contained much higher levels of STAT3 compared to the negative control. In the lower panel of Figure 1, immunoblots stained with anti-FLAG monoclonal antibody revealed the presence of FLAG only in infected cells but not the negative control. The staining intensity of FLAG increased with higher multiplicity of infection (MOI). The increase was most noticeable between MOI of 25 and 50. Immunoblots stained with anti-STAT3 did not show any detectable difference in the intensity of STAT3 staining among different MOI, probably due to its higher immunoreactivity compared with the anti-FLAG antibody. The fact that anti-FLAG only recognized one of the two STAT3 bands may also be related to its weaker immunoreactivity. Similar Western blot results were obtained using lysates from infected SU-DHL-1 cells, indicating that the efficiency of adenoviral infection and

STAT3DN expression are similar between these two ALK þ ALCL cell lines. STAT3DN lacks the Tyr705 residue To demonstrate that STAT3DN lacks the Tyr705 residue, we performed immunoprecipitation studies using antiSTAT3 and an antibody reactive with phosphorylated tyrosine (pTyr). As shown in the upper panel of Figure 2, immunoprecipitation by using anti-STAT3 pulled down more STAT3 than the negative control. The same immunoblot was stained with antibody reactive with pTyr, and the results are shown in the lower panel of Figure 2. The immunoreactivity of pTyr decreased in association with the expression of STAT3DN. These findings confirmed that the STAT3DN construct was mutated at the Tyr705 residue. Cellular localization of STAT3DN According to the current concepts, phosphorylation of the Tyr705 residue of STAT3 is considered to be crucial

Figure 1 Expression of STAT3 at 24 h after infecting Karpas 299 cells with AdSTAT3DN. Compared with the noninfected cells, the levels of STAT3 markedly increased after infection. The lower panel shows that FLAG is only present in the infected cells and not in the negative control

Figure 2 Immunoprecipitation studies for the detection of STAT3 and pSTAT3 in Karpas 299 cells. The upper panel shows that STAT3 expression increased with the infection of AdSTAT3DN, whereas the lower panel shows that pTyr decreased in association with the infection. (WB ¼ Western blot; IP ¼ immunoprecipitation.) Oncogene


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in dimerization and nuclear localization of STAT3. To explore the cellular localization of STAT3DN, we performed Western blot studies after subcellular fractionation and found that STAT3DN was detectable in both the nuclear and cytoplasmic fractions in Karpas 299 cells at 24 h after infection. A slightly higher level of STAT3DN was present in the cytoplasmic fraction (Figure 3). Staining with anti-FLAG showed similar findings. Retinoblastoma protein, which was used as a control, was found only in the nuclear fraction in both the control and treated cells. STAT3DN decreases the viability of ALCL cell lines Following expression of STAT3DN, the biological changes of ALCL cell lines were assessed at 72 h after infection. As shown in Figure 4, the number of viable cells, as assessed by trypan blue exclusion studies, decreased with increasing the MOI. Reduction to 50% of the control was observed at an MOI of 60 for Karpas 299 and at an MOI of 100 for SU-DHL-1. These experiments were performed in triplicate, and the decrease in the number of viable cells was statistically significant compared with the controls (Po0.05, t-test).

Figure 4 Viability of Karpas 299 and SU-DHL-1 cells at 72 h after infection with AdSTAT3DN. Reduction to 50% of the control was detected at an MOI of 50 for Karpas 299 and of 65 for SU-DHL-1. The results represent the means7s.d. of three different experiments. Statistically significant difference was detected between control and treated cells (Po0.05)

Evidence of apoptosis We assessed whether the decrease in viable cells after STAT3DN expression was due to the occurrence of apoptosis by performing flow cytometry studies for the detection of the possible binding of annexin V to the apoptic cell membrane as well as by morphologic evaluation. Increased annexin V uptake was first

detectable at 16 h at MOI that led to 50% reduction in cell viability for both Karpas 299 and SU-DHL-1 cells. As illustrated in Figure 5a, there was concentrationdependent increased binding of annexin V in Karpas 299 cells. The level of annexin V expression correlated with MOI. SU-DHL-1 cells showed similar results. As shown in Figure 5b, morphologic examination of Hoechststained preparations of SU-DHL-1 cells after infection with AdSTAT3DN demonstrated nuclear condensation and fragmentation; supporting the concept that infection with AdSTAT3DN induces apoptotic cell death in these cells. As shown in Figure 5c, Western blot studies revealed the presence of the cleaved products of caspase3, which increased in intensity with higher MOI. Both ALCL cell lines infected with AdGFP at up to MOI of 100 did not show detectable changes with this assay. STAT3DN mediates G1 cell cycle arrest

Figure 3 Subcellular fractionation followed by Western blotting to determine the cellular location of AdSTAT3DN in Karpas 299 cells. The Western blot results show that AdSTAT3DN is present in both the extracts from the cytoplasm and the nucleus. It may be speculated from these findings that nuclear localization of STAT3 is not strictly dependent on the phosphorylation of the Tyr705 residue. Retinoblastoma protein was detected exclusively in the nuclear fraction Oncogene

Cell cycle analysis by flow cytometry using bromodeoxyuridine (BrdU) incorporation and 7-amino-actinomycin D (7-AAD) showed that at 16 h, STAT3DN induced a reduction in the S phase in both cell lines. As illustrated in Figure 6, the proportion of Karpas 299 cells that were reactive with anti-BrdU (i.e. S phase) decreased with increasing MOI. Assessment of SU-DHL-1 cells showed similar results. Both ALCL cell lines infected with AdGFP at up to MOI of 100 did not show detectable changes with this assay (Figure 6). Modulation of downstream targets of STAT3 We examined the possible mechanisms underlying STAT3DN-mediated apoptosis in these two ALCL cell


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Figure 5 (a) Infection of Karpas 299 cells with AdSTAT3DN induced concentration-dependent increase in annexin V expression at 16 h. SU-DHL-1 cells demonstrated similar results. (b) Compared with control SU-DHL-1 cells, Hoechst staining shows nuclear condensation and fragmentation at 24 h after infection with AdSTAT3DN. (c) Cleaved caspase-3 as detected by Western blot in Karpas 299 cells after infection with AdSTAT3DN. Maximum intensity of the band was detected at an MOI of 75

lines. Some of the results obtained from Karpas 299 cells are illustrated in Figure 7. Of the apoptosis-related proteins examined, Bcl-2, Bcl-xL, survivin and Mcl-1 were downregulated. Of the three cyclin D family members, only cyclin D3 was expressed in these two ALCL cell lines. Cyclin D3 was downregulated after infection with AdSTAT3DN. We also examined the expression of c-Myc and suppressors of cytokine signaling (SOCS3), because the expression of these

proteins has been shown to be driven by STAT3 in other cell types (Naka et al., 1997). We found that both c-Myc and SOCS3 were downregulated with increasing MOI of STAT3DN. In contrast, b-actin and cyclindependent kinase 4 (CDK4), both of which are not known to be upregulated by STAT3, had no changes in their protein levels after treatment. In addition, AdGFP did not induce any detectable changes in these proteins. Figure 8 shows a gradual decrease of Mcl-1 protein level Oncogene


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Figure 6 Cell cycle analysis using BrdU uptake and flow cytometry in Karpas 299 cells at 16 h after infection with AdSTAT3DN. The results show notable reduction of the S phase with increasing the MOI of AdSTAT3DN. Infecting the cells with AdGFP at 100 MOI did not induce significant changes in the S phase

over 24 h after gene transfer of STAT3DN, and the decrease in the protein level was first detectable at 18 h. Other downstream targets showed similar pattern of changes in their protein levels after AdSTAT3DN treatment. Discussion The pathogenesis of ALK þ ALCL has been extensively studied in recent years. It is now postulated that the mechanisms of lymphomagenesis involve deregulation of several signaling pathways that may be interacting with each other. Activation of these pathways is triggered by the oncogenic tyrosine kinase, NPMALK. Recently, Zamo et al. (2002) showed that NPM-ALK phosphorylates and activates STAT3, an oncogenic protein. Zhang et al. (2002) showed that STAT3 activation in ALCL may also be attributed to defects involving STAT3-negative regulatory pathways including protein phosphatase 2A and protein inhibitor of activated STAT (PIAS). Regardless of the exact mechanism, STAT3 activation correlates with ALK expression in ALCL tumors (Zamo et al., 2002). While the relationship between STAT3 and ALK is relatively Oncogene

well established, evidence to support the direct pathogenetic role of STAT3 in ALK þ ALCL is lacking. In the present study, we evaluated the importance of STAT3 activation in two ALK þ ALCL cell lines, Karpas 299 and SU-DHL-1, by inhibiting STAT3 signaling using a STAT3DN construct. We also investigated changes in protein expression of a number of potential downstream targets of STAT3. We found that the expression of STAT3DN effectively induces apoptosis and G1 cell-cycle arrest in both ALK þ ALCL cell lines. These findings suggest that STAT3 activation promotes survival and cell cycle progression in these cells. Similar observations have been previously reported in other human cancer types, including myeloma (Catlett-Falcone et al., 1999), breast cancer (Garcia et al., 1997), melanoma (Niu et al., 2002), squamous cell carcinoma (Grandis et al., 1998) and prostatic carcinoma (Mora et al., 2002). STAT3 activation may be an important pathway by which NPMALK mediates oncogenesis. The AdSTAT3DN used in this study has been previously utilized in a number of studies (Negoro et al., 2001; Osugi et al., 2002). The biologic effects of the STAT3DN construct are secondary to a single mutation at residue 705tyrosine-phenylalanine. This mutation


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Figure 7 Western blot studies showing changes in apoptotic and cell cycle related proteins in Karpas 299 after being infected with increasing MOI of AdSTAT3DN. There was progressive decrease in Bcl-2, Bcl-xL, SOCS3, survivin, Mcl-1, cyclin D3 and c-Myc. b-actin levels were comparable among all samples, as shown in the lower panel. In addition to b-actin, the STAT3DN-induced changes were not seen in another protein not regulated by STAT3; the CDK4 (data not shown)

Figure 8 STAT3DN induces time-dependent decrease in Mcl-1. The effect of STAT3DN became more pronounced at 18 h after infecting Karpas 299 cells

prevents tyrosine phosphorylation at this site, which is believed to be crucial in mediating dimerization and nuclear localization of STAT3 (Bromberg et al., 1999; Bromberg and Darnell, 2000). Our findings from immunoprecipitation studies support this concept, since STAT3DN did not show evidence of tyrosine phosphorylation in Karpas 299 cells. The exact mechanism underlying the dominant-negative effects of this construct is not clear. Nevertheless, our immunoprecipitation studies showed that expression of STAT3DN induces downregulation of tyrosine phosphorylation of the endogenously expressed STAT3. Since phosphorylation of the Tyr705 residue of STAT3 is believed to be important for dimerization and nuclear localization of STAT3, we assessed the cellular localization of STAT3DN. To our surprise, subcellular fractio-

nation studies showed that approximately half of the expressed STAT3DN protein is localized into the nucleus. These findings indicate that a yet to be defined mechanism exists to regulate the trafficking of STAT3 between the cytoplasm and nucleus. It is of note that a recent study demonstrated that arginine/lysine-rich regions, rather than tyrosine residues, are involved in the nuclear localization signals (Fagerlund et al., 2002). It is likely that STAT3 tyrosine phosphorylation and dimerization are regulated differently from nuclear localization of STAT3. Further studies are warranted to investigate the regulatory mechanism of STAT3 trafficking. The present study shows that STAT3DN induces G1 cell cycle arrest. Cyclin D3 is highly expressed in ALK þ ALCL cells, and recently cyclin D genes have been found to have a STAT3 binding consensus sequence in their promoters (Matsui et al., 2002). Another G1 cell cycle regulator, c-Myc, also contains a STAT3 binding consensus sequence in its promoter. Our results showed that both cyclin D3 and c-Myc are effectively downregulated by the expression of STAT3DN. In view of the normal biologic role of cyclin D genes and c-Myc as positive regulators of G1 cell cycle progression, it is likely that downregulation of cyclin D3 and c-Myc contributes to the G1 cell cycle arrest seen in both ALK þ ALCL cell lines after the expression of STAT3DN. In this study, we also found that STAT3DN induces apoptosis in both ALK þ ALCL cell lines. We observed the most dramatic decrease in the protein levels of the antiapoptotic proteins survivin and Mcl-1. Aoki et al. (2002) showed that inhibition of STAT3 signaling induces apoptosis via downregulation of survivin without modulating the antiapoptotic Bcl-2 family members including Bcl-2, Bcl-xL and Mcl-1. Our findings are similar to those of this study in that STAT3DN-induced apoptosis was associated with the downregulation of survivin. Survivin has been previously shown to be an important antiapoptotic protein (Altieri, 2001). Mahboubi et al. (2001) showed that survivin induces IL-11mediated cell survival through a STAT3 pathway. Aoki et al. (2002) also showed that enforced expression of survivin suppresses cell death induced by STAT3 inhibition. It is likely that survivin contributes to STAT3-mediated tumorigenetic effects by promoting cell survival. Although expression of survivin in various types of lymphoma has not been comprehensively studied, it appears to be expressed in lymphoma types that have constitutive STAT3 activation, including Hodgkin lymphoma (Kube et al., 2001), primary effusion lymphoma (Aoki et al., 2002), as well as ALCL (Schlette et al., in press). These findings are in keeping with the concept that STAT3 drives the transcription of survivin. In contrast with previously published studies in other types of lymphoma (Aoki et al., 2002), we observed a significant reduction in the expression of another antiapoptotic protein, Mcl-1, after STAT3DN treatment. Mcl-1 was found to be overexpressed in ALK þ ALCL cell lines and tumors, which further supports our Oncogene


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findings (Rassidakis et al., 2002). The relevance of individual downstream targets of STAT3 appears to be cell-type specific. In other lymphoid cell types, a link between STAT3 activation and regulation of Bcl-2 family of proteins has been demonstrated (CatlettFalcone et al., 1999; Nielsen et al., 1999; EplingBurnette et al., 2001). In keeping with previous studies (Catlett-Falcone et al., 1999), gene transfer of STAT3DN into ALK þ ALCL in our study induced a decrease in protein expression of Bcl-2 and Bcl-xL. SOCS3, a negative regulator of STAT3, was downregulated when STAT3DN was expressed. The SOCS proteins comprise a family of negative regulators of cytokine signaling, each characterized by a central SH2 domain and a C-terminal SOCS box. SOCS3, one of eight members of this family, is able to suppress signaling induced by an array of cytokines including IL-2, IL-4, IL-6, IFN-g, IFN-a, leukemia inhibitory factor and growth hormone (Alexander et al., 1999). SOCS3 has been shown to bind directly to the kinase domain of JAK kinases and inhibit their activity by acting as a pseudosubstrate (Sasaki et al., 1999). Normally, transcription of SOCS genes is induced after stimulation with cytokines, and the expression of SOCS is generally short lived (Hilton, 1999). Previous studies have suggested that STATs are important in regulating the expression of SOCS proteins, and STAT binding sites have been identified in the SOCS3 promoter (Naka et al., 1997). We found that SOCS3 is highly expressed in both ALK þ ALCL cell lines. In addition, our finding that the expression of STAT3DN effectively downregulated SOCS3 strongly suggests that the relatively high expression of SOCS3 in these cells is dependent on the presence of activated STAT3. Similar findings also have been identified in T-cell lymphoma cell lines (Brender et al., 2001). Since SOCS3 mediates the suppression of a wide range of cytokines, the relatively high level of SOCS3 expression in ALK þ ALCL cells may contribute to the augmentation of the effects of a number of cytokines, leading to the escape of the normal regulatory growth and differentiation signals. Results from the present study suggest that direct inhibition of STAT3 using gene therapy approach may represent a promising modality for the treatment of ALK þ ALCL. This concept is in agreement with several earlier findings showing that activated STAT3 signaling contributes to the growth and survival of diverse human cancer cells (Garcia et al., 1997; Grandis et al., 1998; Catlett-Falcone et al., 1999; Mora et al., 2002; Niu et al., 2002). Currently, investigators are developing various pharmacologic inhibitors of STAT3 that can be utilized for cancer therapy (Turkson and Jove, 2000; Buttner et al., 2002). In summary, by using STAT3DN, we have provided further evidence that STAT3 activation contributes to cell proliferation and survival in ALK þ ALCL cells. We have also identified a number of downstream targets of STAT3 that might be directly involved in the pathogenesis of ALK þ ALCL, although the relative importance of these target proteins needs to be further elucidated. Further studies addressing STAT3 signaling and its Oncogene

downstream targets may provide important information useful for developing new therapeutic agents for various types of human cancer, including ALK þ ALCL.

Materials and methods ALCL cell lines Two ALK þ ALCL cell lines, SU-DHL-1 and Karpas 299, were used in this study. Both cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Germany) and have been described previously (Epstein and Kaplan, 1979; Wood et al., 1996). The cell lines were grown in 10% Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum, 10 000 U/ml penicillin (Sigma, St Louis, MO, USA), 10 mg/ml streptomycin (Sigma) and 29.2 mg/ml L-glutamine (Life Technologies), and incubated at 371C in 95% air and 5% CO2. STAT3DN adenoviral vector The production and characteristics of AdSTAT3DN have been detailed previously (Kunisada et al., 1998). Briefly, the adenoviral vector was deleted at the E1A region. Using the site-directed mutagenesis technique, the STAT3 cDNA was modified such that the Tyr705 residue was replaced by phenylalanine. Expression of STAT3 was driven by the rabbit b-actin promoter and cytomegalovirus enhancer. The construct was epitope-tagged with FLAG (DYKDDDDK) (Kodak) at the N-terminal. Antibodies Monoclonal antibodies specific for the following proteins were used in this study: cyclin D3, STAT3, c-Myc, Mcl-1, pTyr (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl-2 (Dako, Carpinteria, CA, USA), Bcl-xL (Zymed, San Francisco, CA, USA), cleaved products of caspase-3 (BD Biosciences, San Diego, CA, USA), survivin (Novus Biologicals, Littleton, CO, USA), b-actin (Sigma, St Louis, MO, USA) and FLAG (Abcam, Cambridge, UK). Detection of apoptosis (a) Morphologic changes: after infection with adenoviral vectors at different MOI, Cytospin cell preparations (100 ml at a concentration of 1.0 106 cells/ml) were incubated with 1 ml of Hoechst 33258 (1 mg/ml in distilled water) for 10 min at room temperature. Subsequently, cells were examined under a fluorescence microscope. Cells were considered to be apoptotic when the nuclei showed chromatin condensation and fragmentation. (b) Flow cytometry using annexin V/7AAD staining was performed according to the manufacturer’s guidelines. Briefly, 0.5 106 cells were washed in ice-cold phosphate-buffered saline (PBS) without Ca2 þ or Mg2 þ (Life Technologies). The cells were then resuspended in 100 ml of binding buffer and incubated with 5.0 ml of 7-AAD and 2.0 ml of annexin V-PE for 15 min in the dark at room temperature. Flow cytometric analysis was immediately performed using a flow cytometer (Becton Dickinson, San Jose, CA, USA). Cell cycle analysis by flow cytometry Cell cycle analysis was performed using a BrdU kit (BD Biosciences) and analysed using a flow cytometer in accordance


5433 with the manufacturer’s recommended protocol. Briefly, ALCL cells were incubated with BrdU at a final concentration of 10 mM in a cell culture medium for 1 h at 371C. After washing in cold PBS, cells were fixed with 100 ml Cytofix/Cytoperm buffer for 15–30 min on ice, and then treated with DNase for 1 h at 371C. After washing, FITC-labeled anti-BrdU antibody was incubated with the cells for 20 min at room temperature. For AdGFP-infected cells, PE-labeled anti-BrdU antibody was used instead. After washing, the cells were suspended with 7AAD and analysed by flow cytometry.

Immunoprecipitation and tyrosine phosphorylation status Tyrosine phosphorylation of STAT3 was evaluated using immunoprecipitation. Cell lysates prepared as above were incubated with anti-STAT3 antibody overnight at 41C and subsequently with protein A/G sepharose (Santa Cruz) for 4 h at 41C. Four washes with cold PBS and one wash with lysis buffer were then performed, followed by boiling with 25 ml of SDS– PAGE loading buffer (62.5 mM, Tris, pH 6.8, 2% SDS, 5% 2mercaptoethanol and 10% glycerol) for 5 min. Thereafter, the samples were centrifuged at 2000 g for 3 min and the supernatants were collected and stored at 801C until the time of assay.

Western blot analysis Western blot analysis was performed using standard techniques. Briefly, the cells were washed in PBS (pH ¼ 7.5) and lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% sodium dodecylsulfate (SDS), 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 5 mg/ml aprotonin (5 mg/ ml), sodium vanadate (1 mM) and leupeptin (5 mg/ml). After incubation on ice for 15 min, the lysates were subjected to centrifugation at 12 000 r.p.m., and the supernatants were collected. Protein concentration was determined with a protein assay kit (Bio-Rad, Hercules, CA, USA). Each lane of a 6 or 12% polyacrylamide gel (PAGE) was loaded with 80 mg of protein. After electrophoresis and transfer to nitrocellulose membranes (Bio-Rad) by electroblotting, blots were probed with specific primary and secondary antibodies and the enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL, USA) according to the manufacturer’s protocol. The antibodies were used at dilutions of 1 : 500 to 1 : 1000.

Subcellular fractionation Subcellular fractionation was achieved using the NE-PERt nuclear and cytoplasmic extraction kit (Pierce, Rockford, IL, USA) as per the manufacturer’s protocol with minor modifications. Briefly, 20 ml of packed cells infected with AdSTAT3DN or AdGFP were washed twice with cold PBS and centrifuged. The cell pellet was treated with 200 ml of cytoplasmic extraction reagent on ice for 10 min, with gentle mixing periodically. Subsequently, 11 ml of cytoplasmic extraction reagent II was added to the mixture and incubated on ice for 1 min. The mixture was subjected to centrifugation at 16 000 g and the supernatant (the cytoplasmic extract) was transferred to a clean prechilled tube, and stored at 801C until use. The pellet was resuspended in 100 ml of ice-cold nuclear extraction reagent, vortexed for 15 s every 10 min, for a total of 40 min. The mixture was then centrifuged at 16 000 g for 10 min, and the supernatant (nuclear extract) was transferred to a prechilled tube, and stored at 801C until use.

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