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[Cancer Research 65, 8224-8232, September 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Disruption of the Inhibitor of Apoptosis Protein Survivin Sensitizes Bcr-abl–Positive Cells to STI571-Induced Apoptosis

Zhanxiang Wang, Janardhan Sampath, Seiji Fukuda and Louis M. Pelus

Department of Microbiology and Immunology and the Walther Oncology Center, Indiana University School of Medicine, and the Walther Cancer Institute, Indianapolis, Indiana

Requests for reprints: Louis M. Pelus, Department of Microbiology and Immunology and the Walther Oncology Center, Indiana University School of Medicine, 950 West Walnut Street, Indianapolis, IN 46202. Phone: 317-274-7565; Fax: 317-274-7592; E-mail: lpelus{at}iupui.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Bcr-abl oncogene induces hematopoietic cell transformation and protects cells from apoptosis; however, the mechanisms whereby Bcr-abl blocks apoptosis are poorly defined. We examined whether the inhibitor of apoptosis protein (IAP) family, in particular survivin, are regulated by Bcr-abl. Overexpression of Bcr-abl in Mo7e or BaF3 hematopoietic cells elevated survivin mRNA and protein concomitant with a 4-fold increase in survivin promoter activity. The region of the survivin promoter responding to Bcr-abl was narrowed down to a 116 bp fragment between nucleotides –1,194 and –1,078. The IAP family member IAP-like protein-2 was also up-regulated by Bcr-abl. Disruption of Bcr-abl in Bcr-abl–transduced BaF3 cells by small interfering RNA resulted in 3- to 4-fold reduction in survivin protein confirming the link between Bcr-abl and survivin. Survivin disruption in Bcr-abl–transduced Mo7e cells, or in K562 cells that endogenously express Bcr-abl, by transfection with dominant-negative or antisense survivin constructs promoted apoptosis induced by the Bcr-abl tyrosine kinase inhibitor STI571, which was accompanied by caspase-dependent cleavage of Bcr-abl, mitochondrial membrane potential disruption, and enhanced mitochondrial cytochrome c release. Although ectopic survivin protected K562 cells from apoptosis induced by STI571, it did not protect cells from apoptosis induced either by tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) or the combination of TRAIL plus Hemin. Our results identify a new signal pathway downstream of Bcr-abl, in addition to the Bcl-2 family involved in the antiapoptotic effects of Bcr-abl, and suggest that anti-survivin therapy may have utility in patients with chronic myelogenous leukemia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Bcr-abl oncogene, a result of a reciprocal translocation between chromosomes 9 and 22 [t(9;22)], is found in the cells of 95% of patients with chronic myelogenous leukemia (CML; ref. 1) and encodes a cytoplasmic protein with constitutive kinase activity (2). Bcr-abl induces hematopoietic cell transformation (3) and protects cells from apoptosis induced by numerous stimuli (4). Critical signals for Bcr-abl transforming activity are mediated by downstream signaling pathways normally activated by receptor tyrosine kinases; however, the mechanisms responsible for its antiapoptotic effects and the signaling pathways connecting Bcr-abl to the apoptosis death machinery are poorly defined.

The Bcl-2 and inhibitor of apoptosis protein (IAP) families regulate apoptosis (5). Bcr-abl can exert an antiapoptotic effect by blocking mitochondrial release of cytochrome c, via the Bcl-2 family, because high Bcr-abl expression prevents their early translocation to the mitochondria, preserves mitochondrial membrane potential, and blocks caspase activation (6). However, Bcr-abl has a much stronger antiapoptotic effect than Bcl-xL, suggesting that additional/alternative survival pathways are involved (7). The IAP survivin blocks apoptosis by inhibiting caspases (8) and is aberrantly expressed in almost all cancers and hematopoietic malignancies, which correlates with poor prognosis (9). Targeted disruption of survivin in several transformed cell models enhances apoptosis (10, 11).

Survivin is highly expressed during blast crisis in patients with Bcr-ablPos, but not in patients with Bcr-ablNeg CML (12), and elevated survivin is found in Adriamycin-resistant Bcr-ablPos K562 cells (13). These findings raise the questions of whether survivin is involved in the antiapoptotic effects of Bcr-abl and if survivin disruption can promote apoptosis in Bcr-ablPos cells. We examined whether IAPs, in general, and survivin, in particular, are regulated by Bcr-abl. Our results show that Bcr-abl elevates survivin and IAP-like protein-2 (ILP-2) mRNA and protein and enhances survivin promoter activity. Moreover, survivin disruption sensitizes Bcr-ablPos cells to apoptosis induced by the Bcr-abl tyrosine kinase inhibitor STI571, as well as to apoptosis induced by chemotherapeutic agents through the intrinsic mitochondrial pathway. In contrast, survivin disruption does not protect cells from apoptosis initiated via the extrinsic pathway. Our results identify a new signaling pathway downstream of Bcr-abl involved in its antiapoptotic effects and suggest that anti-survivin strategies may have therapeutic utility in patients with CML.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies. Anti-human survivin (AF886) was purchased from R&D Systems (Minneapolis, MN). Anti–c-abl (Ab-3) was from EMD Biosciences (La Jolla, CA). Anti-phosphotyrosine, tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL), Hemin, cis-diammine platinum dichloride (CDDP), cytosine arabinofuranoside (Ara-C), and 3,3'-diethyloxadicarbocyanine iodide (DODCB) were purchased from Sigma Chemical Company (St. Louis, MO). Goat anti-actin was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti–ILP-2 was purchased from ProSci, Inc. (Poway, CA). The broad-spectrum caspase inhibitor z-VAD-fmk was from Apotech (San Diego, CA). STI571 (Gleevec) was obtained from Novartis, Inc. (Basel, Switzerland).

Cell lines, plasmids, antisense oligonucleotides, and transfection. The Mo7e leukemia cell line has been described (14). Bcr-abl–transduced Mo7e cells were obtained from Dr. Scott Boswell. Mouse BaF3 cells stably transduced with Bcr-abl or control vector were provided by Dr. M.W. Deininger. Full-length, antisense, and T34A human survivin cloned in the IRES-EGFP-MIEG3 vector were prepared as described (15). The C84A survivin (16) construct was generated using the primers 5'-phospho-TTGGCCTGCTTACGAAAA-3' and 5'-phospho-CGGCGAAAGGAAAGACAG-3' (15). Log-phase Mo7e and K562 cells plus 30 µg of survivin plasmid DNAs were mixed in a Bio-Rad gene Pulser Cuvette and incubated at 37°C for 15 minutes. Following one pulse electroporation (360 V, 960 µF), cells were incubated at 37°C for 15 minutes and resuspended in RPMI 1640 + 10% heat-inactivated fetal calf serum (FCS) with 10 ng/mL recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF). After 48 hours, green fluorescent protein–positive cells were isolated by fluorescence-activated cell sorting (Becton Dickinson, San Jose, CA), expanded for 1 week, and resorted for green fluorescent protein–positive cells. A 20-mer phosphorothioate antisense oligonucleotide targeting survivin mRNA, 5'-C*C*C*AGCCTTCCAGCTCCT*T*G*-3' (17), and a random nonsense control oligo, 5'-C*A*C*CGCCTCTCATCGTCG*T*C*-3', were purchased from Integrated DNA Technologies (Coralville, IA). Cells were incubated with 60 µmol/L anti-survivin oligos and electroporation carried out as described.

RNA isolation and reverse transcription-PCR. RNA isolation and reverse transcription-PCR (RT-PCR) amplification of IAPs were carried out as described (18). PCR primers for survivin, Livin, XIAP, cellular IAP 1 (cIAP1), cIAP2, neuronal AIP (NAIP), and Apollon were as previously reported (18). Reverse primers for survivin-2B and {Delta}Ex3 were 5'-GTGCTGGTATTACAGGCGTAAG-3' and 5'-TGGTTTCCTTTGCATGGGG-3', respectively. The forward primer was 5'-GGCGGCATGGGTGCCCCGACGTT-3'. The human ILP-2 primers were 5'-ACTTGAGGGAGCTCTGGTACAAAC-3' and 5'-AGTGACCAGATGTCCACAAGC-3'. The mouse survivin primers were 5'-TGGCAGCTGTACCTCAAGAA-3' and 5'-AGCTGCTCAATTGACTGACG-3'.

Quantitative real-time reverse transcription-PCR. Primers and probes for human WT survivin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using Primer Express Software (Applied Biosystems, Foster City, CA) and purchased from Applied Biosystems. Primers and probes for survivin were previously described (18). Survivin-2B and survivin-{Delta}Ex3 were quantitated using Platinum SYBR Green UDG Supermix Kit (Invitrogen, Carlsbad, CA) using the same primers used for RT-PCR. Total RNA was treated with DNase (Promega, Madison, WI) for 30 minutes at 37°C, followed by real-time RT-PCR. The RT-PCR conditions and calculation according to the {Delta}CT value were as described (18).

Subcellular fractionation, cytoplasmic and nuclear extraction, and Western blot analysis. Cell cytosolic and mitochondrial fractions were prepared using the Cytochrome C Releasing Apoptosis Assay Kit (Biovision, Mountain view, CA). Cytoplasmic and nuclear extractions were done using NE-PER (Pierce Biotechnology, Rockford, IL). Cell lysates for Western blots were prepared in buffer containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µg/mL phenylmethylsulfonyl fluoride, and 1 mmol/L sodium orthovanadate. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Fifty micrograms of protein were denatured in 2x SDS buffer at 95°C for 5 minutes, separated on 10% SDS-PAGE gels (Invitrogen), and transferred onto nitrocellulose membranes. Membranes were incubated with specific antibodies followed by horseradish peroxidase–conjugated anti-mouse or anti-rabbit immunoglobulin, and signal was detected by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). Bcr-abl and c-abl cleavage was determined using anti–c-abl antibody.

Bcr-abl knockdown using small interfering RNA. BaF3-bcr-abl cells were mixed with 5 µg of Bcr-abl–specific small interfering RNA (siRNA; ref. 19) or nonsilencing control siRNA (Qiagen, Valencia, CA), incubated at 37°C for 15 minutes, and electroporated at 280 V and 960 µF. Following incubation at 37°C for 15 minutes, cells were resuspended in RPMI 1640 with 10% heat-inactivated FCS. Cells were harvested 72 hours post electroporation, washed with PBS plus 1 mmol/L sodium orthovanadate, and lysed in radioimmunoprecipitation assay buffer [20 mmol/L Tris-HCl (pH 8.0), 5 mmol/L EDTA, 150 mmol/L NaCl, 10 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 1% NP40, 1% sodium deoxycholate, 2 mmol/L sodium orthovanadate] containing protease inhibitor cocktail (Roche, Indianapolis, IN) and analyzed by Western blot.

Caspase-9 activity assay. Caspase-9 activity in cytoplasmic extracts was determined using a 96-well format Caspase-9 Colorimetric Assay Kit according to the instruction of the manufacturer (Biovision). Caspase-9 activity was expressed as absorbance at 450 nm.

Survivin promoter luciferase assays. Vector- and Bcr-abl–transduced Mo7e cells were transfected with human survivin promoter [nucleotides (nt) –1,895 to +366] in the pGL3 Luciferase Reporter Vector, obtained from Dr. S. Liu (Schering-Plough Research Institute, Kenilworth, NJ). To generate a minimal promoter construct (–71 to +366), PCR was carried out using the primers 5'-ACAGAGCTCCCAGAAGGCCGCGGGGG-3' and 5'-ACAAAGCTTAGCCACAAAGGCCTCGATGGG-3' using the full-length promoter (–1,895 to +366) as template (20). The PCR product was gel purified, digested with SacI and HindIII, and ligated into the SacI/HindIII site of pGL3 Luciferase Reporter Vector (Promega). To identify Bcr-abl regulated region(s) in the survivin promoter, site-directed mutagenesis and specific promoter deletions were constructed using the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using the full-length survivin promoter as template. The primer pairs used were as follows: GATA-WTMT1, 5'-CCTGCCAAAGTCAACTGGATTCATATGCTGCAGCGAAGTTAAG-3' and 5'-CTTAACTTCGCTGCAGCATATGAATCCAGTTGACTTTGCCAGG; {Delta}–1,494 to –1,387WT{Delta}2, 5'-GCAGAGAGTGAATGTTCAGTGGCTCACATCTG-3' and 5'-CAGATGTGAGCCACTGAACATTCACTCTCTGC-3'; {Delta}–1,194 to –1,078WT{Delta}3, 5'-GCAGGAGAATCGCTTCTGTATTAAAGAATGGGG-3' and 5'-CCCCATTCTTTAATACAGAAGCGATTCTCCTGC-3'; {Delta}–980 to –902WT{Delta}4, 5'-GAAAAAGACAGTGGAGATGTGATGCCCAGC-3' and 5'-GCTGGGCATCACATCTCCACTGTCTTTTTC-3'; {Delta}–800 to –681WT{Delta}5, 5'-GACTTACTGTTGGTGGTGACTCCAGAAGGTG-3' and 5'-CACCTTCTGGAGTCACCACCAACAGTAAGTC-3'; {Delta}–399 to –295WT{Delta}6, 5'-CAAGCGATTCTCCTGTCTTGAACTCCAGG-3' and 5'-CCTGGAGTTCAAGACAGGAGAATCGCTTG-3'. Plasmid DNA sequence was obtained to verify the intended mutation/deletion and absence of random mutations. Twenty micrograms of survivin promoter firefly luciferase construct and 1 µg of Renilla plasmid were electroporated into Mo7e cells. Eighteen hours after transfection, cell lysates were prepared using the Luciferase Assay System Kit (Promega). Dual luciferase activity was measured with a DLReady Femtomaster FB12 Luminometer (Zylux Corp., Maryland, TN).

Analysis of mitochondrial membrane potential. Mitochondrial membrane potential ({Delta}{Psi}m) was measured by flow cytometry using the fluorescent cationic dye tetramethylrhodamine methyl ester (TMRM) or 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1; Sigma; refs. 21, 22). For TMRM staining, cells were washed in PBS, resuspended in 50 µL HEPES buffer (10 mmol/L HEPES, 135 mmol/L NaCl, 5 mmol/L CaCl2), and incubated with 200 nmol/L TMRM for 15 minutes at 37°C. For JC-1 staining, cells were incubated with JC-1 for 15 minutes at 37°C and fluorescence was measured in both the FL-1 and FL-2 channels with an excitation of 488 nm. JC-1 is a dual emission probe with increased fluorescence at 530 nm (FL-1) and reduced fluorescence at 590 nm (FL-2) associated with a decrease in {Delta}{Psi}m.

Flow cytometry analysis of apoptosis. To quantitate hypodiploid DNA content, cells were fixed overnight in 70% ethanol at 4°C, washed with PBS, and resuspended in 0.5 mL of propidium iodide buffer (50 µg/mL propidium iodide in PBS) for 30 minutes. DNA analysis was done by flow cytometry as previously described (15).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Survivin mRNA and protein are up-regulated in Bcr-abl–transduced Mo7e and BaF3 cells. Because survivin is overexpressed in Bcr-ablPos CML cells (12) and Adriamycin-resistant Bcr-ablPos K562 cells (13), we asked whether survivin expression is regulated by Bcr-abl. RT-PCR indicated that mRNA for WT survivin and the splice variants {Delta}Ex3 and 2B (23) were detected in control vector–transduced Mo7e cells and elevated in Bcr-abl–transduced cells (Fig. 1A, B, and D). Quantitative RT-PCR showed an equivalent 2- to 3-fold increase in each of the survivin transcripts compared with controls (Fig. 1A). Full-length survivin was the dominant transcript detected in control- and Bcr-abl–transduced Mo7e cells, and survivin-{Delta}Ex3 levels were higher than survivin-2B. Total survivin protein was elevated by ~3-fold in Bcr-abl–transduced cells (Fig. 1B and C). Similar to Mo7e cells, survivin mRNA and protein were up-regulated in BaF3 cells transduced with human Bcr-abl (Fig. 1C). To determine if survivin up-regulation was a consequence of Bcr-abl overexpression, we electroporated BaF3 cells stably expressing Bcr-abl with Bcr-abl–specific (19) or control siRNA. Western blots at 72 hours post electroporation (Fig. 1E) clearly showed reduced Bcr-abl and survivin levels in BaF3-bcr-abl cells electroporated with Bcr-abl–specific siRNA. Quantitation of the Bcr-abl knockdown showed a ~1.5- to 2-fold reduction in Bcr-abl protein with a 3- to 4-fold reduction in survivin protein. These results support the fact that elevated production of survivin in Bcr-abl–transduced cells is a consequence of elevated Bcr-abl.



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  		Figure 1. Survivin mRNA and protein are up-regulated in Bcr-abl–transduced Mo7e and BaF3 cells. A, full-length survivin, survivin-{Delta}Ex3, and survivin-2B mRNA were quantitated by real-time RT-PCR in log-phase Mo7e cells transduced with vector or stably expressing human Bcr-abl. Data are expressed as fold change in mRNA levels in three separate experiments. *, P < 0.05. B, Bcr-abl and survivin protein levels in parental and Bcr-abl–transduced Mo7e cells were quantitated by Western blot analysis. Representative Western blots and relative density of survivin and ß-actin from three experiments as determined by densitometry analysis. *, P < 0.05. C, Bcr-abl protein and survivin mRNA and protein were determined in control vector– and Bcr-abl–transduced mouse BaF3 cells. Representative of three experiments. D, RNA was isolated from log-phase vector control– and Bcr-abl–transduced Mo7e cells and RT-PCR was done with primers specific for each of the eight known IAP family members and GAPDH. Representative of three separate experiments. Livin was not detected. Whole-cell lysates from log-phase vector- and Bcr-abl–transduced Mo7e cells were subjected to Western blot analysis for ILP-2 and ß-actin. Data are from one of two experiments with similar results. E, Western blot for Bcr-abl and survivin in BaF3-bcr-abl cells at 72 hours post electroporation with control (–) or Bcr-abl–specific siRNA (+). Data are from one of two experiments with similar results.

 
In addition to survivin, Apollon, cIAP1, cIAP2, NAIP, and ILP-2 were detected by RT-PCR in control vector– and Bcr-abl–transduced Mo7e cells; however, ILP-2 mRNA was the only other IAP elevated in Bcr-abl–transduced cells (Fig. 1D), suggesting that up-regulation is not a general consequence of Bcr-abl transduction. Coincident with elevated mRNA, ILP-2 protein was also elevated. The IAP Livin was not detected, which is similar to our finding in primary hematopoietic cells (18).

We evaluated survivin promoter activity in Bcr-abl–transduced Mo7e cells to determine the nature of enhanced survivin production. Survivin promoter activity was ~4-fold higher in Bcr-abl–transduced cells compared with control cells using luciferase reporter constructs containing a single copy of the WT survivin promoter (–1,895 to +366; Fig. 2A). Enhanced promoter activity was absent in Bcr-abl–transduced Mo7e cells transfected with a minimal survivin promoter (–71 to +366), indicating that the effects of Bcr-abl were manifested on the –1,824 nt upstream region. To further characterize the Bcr-abl responsive region, we deleted defined regions of the survivin promoter and created a reporter plasmid with a mutation in a putative GATA binding site, based on a report that Bcr-abl induces transcription of heat shock protein 70 through a GATA response element (Fig. 2B; ref. 24). The reporter constructs were electroporated into control- and Bcr-abl–transduced Mo7e cells and luciferase activity compared with the full-length WT promoter in each cell line. A significant 77 ± 1% decrease in promoter activity was observed in Mo7e-bcr-abl cells transfected with the {Delta}–1,194 to –1,078WT{Delta}3 deletion mutant (Fig. 2B), with no significant reduction in activity observed with any of the other constructs, suggesting that the effect of Bcr-abl on activation of survivin expression is mediated via transcription factor binding sites present between nt –1,194 and –1,078. The {Delta}–1,194 to –1,078WT{Delta}3 reporter construct also reduced luciferase activity in control cells, but to a lesser degree (33 ± 1%). The {Delta}–399 to –295WT{Delta}6 mutant enhanced activity in Mo7e-bcr-abl and control cells, suggesting the presence of repressor binding sites.



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  		Figure 2. Survivin promoter activity is increased in Bcr-abl–transduced Mo7e cells. A, control vector– and Bcr-abl–transduced Mo7e cells were transiently cotransfected with Renilla luciferase plasmid and firefly luciferase survivin promoter (–1,895 to +366) or truncated (–71 to +366) survivin promoter. Firefly luciferase values were normalized to Renilla luciferase values. Columns, mean relative luminescence from three experiments; bars, SE. *, P < 0.001. B, firefly luciferase constructs with GATA binding site mutation and deletions created from the full-length WT survivin promoter (–1,895 to +366) were electroporated into control Mo7e and Mo7e-bcr-abl cells along with Renilla luciferase reporter. Inhibition of firefly luciferase activity in deletion and GATA mutants is expressed as percent inhibition relative to the full-length WT promoter in each cell line (columns, mean; bars, SE); data from six experiments.

 
Because survivin promoter activity can be regulated in a cell cycle–dependent manner (25), we analyzed cell cycle distribution of Bcr-abl– and vector-transduced Mo7e cells before harvest for the luciferase reporter assay. Fewer Bcr-abl–transduced Mo7e cells were in S and G2-M phases compared with control transduced cells, ruling out the possibility that enhanced survivin promoter activity was related to cell cycle (not shown).

Survivin disruption sensitizes Bcr-ablPos cell lines to apoptosis induced by STI571, cis-diammine platinum dichloride, cytosine arabinofuranoside, and 3,3'-diethyloxadicarbocyanine iodide, but not by tumor necrosis factor–related apoptosis-inducing ligand. The Bcr-abl tyrosine kinase can be selectively inhibited by STI571 (26). We have previously shown that survivin disruption promotes apoptosis in hematopoietic cells (18, 27, 15). Because Bcr-abl up-regulates survivin expression, we hypothesized that survivin disruption would promote STI571-induced apoptosis. In Mo7e-bcr-abl cells, transient overexpression of T34A and C84A dominant-negative or antisense survivin constructs sensitized cells to STI571-induced apoptosis, measured by hypodiploid DNA content (Fig. 3A) and disruption of mitochondrial membrane potential ({Delta}{Psi}m; Fig. 3A, inset). Ectopic survivin protected cells from STI571-induced apoptosis. Similar effects were observed in K562 cells, which express endogenous Bcr-abl (Fig. 3B). Enhanced apoptosis induced by STI571 was observed for at least 72 hours posttransfection with T34A, C84A, or antisense survivin (Fig. 3C). Western blot analysis showed significantly reduced, although incomplete, reduction in survivin protein in cells transfected with antisense survivin (Fig. 3C, inset). Survivin disruption by the antisense construct also enhanced apoptosis induced by the chemotherapeutic agents CDDP and Ara-C (Fig. 3D), which induce apoptosis through the intrinsic pathway (28, 29). The telomerase inhibitor DODCB had an apoptosis-inducing effect on K562 cells, which was enhanced by survivin disruption.



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  		Figure 3. Disruption of survivin sensitized Bcr-abl–transfected Mo7e cells and Bcr-ablPos K562 cells to apoptosis induced by STI571, but not by TRAIL. Bcr-abl–transduced Mo7e cells were transfected with control vector, full-length WT survivin, dominant-negative T34A survivin, dominant-negative C84A survivin, or antisense survivin, and cultured with or without STI571 for 48 hours. A, apoptosis was evaluated by quantitating hypodiploid DNA content (sub-G1) and mitochondrial membrane potential disruption (inset). *, P < 0.001 (data from three experiments). Inset, representative survivin protein expression in control and transfected Mo7e-bcr-abl cells. Survivin protein was detected using polyclonal anti-human survivin. B, hypodiploid DNA content (sub-G1) was evaluated in K562 cells expressing full-length WT, T34A, C84A, and antisense survivin cultured with increasing doses of STI571. Data are from three separate experiments with similar results. Inset, representative survivin protein expression in control and transfected Mo7e-bcr-abl cells. C, apoptosis (sub-G1) in K562 cells following 48 and 72 hours of culture with 0.5 µmol/L STI571. Data are from three separate experiments with similar results. *, P < 0.05. Inset, survivin protein in K562 cells transfected with the antisense survivin is shown by Western blot. D, sub-G1 content in control vector– and antisense survivin–transfected K562 cells cultured for 24 and 48 hours in the presence of 30 µmol/L CDDP, 20 µmol/L Ara-C, or 0.25 µmol/L DODCB. Data are from three to five experiments. *, P < 0.05, compared with vector control cells. E, K562 cells expressing WT survivin, antisense survivin, or control vector were cultured with 20 µmol/L Hemin, 100 ng/mL TRAIL, or the combination of both for 20 hours, and hypodiploid DNA was quantitated by flow cytometry. Data are from three experiments. *, P < 0.001.

 
K562 cells are resistant to TNF{alpha}, Fas ligand, and TRAIL, which induce apoptosis via death receptor pathways (30, 31). In addition, survivin partially inhibits Fas-induced apoptosis (32). To rule out that enhanced apoptosis on survivin disruption was nonselective, we evaluated the effect of transient antisense survivin transfection on apoptosis induced through the death receptors. Vector-transfected K562 cells were resistant to 100 ng/mL TRAIL but sensitive to apoptosis induced by TRAIL plus the erythroid-differentiating compound Hemin (Fig. 3E), consistent with a previous report (30). Ectopic antisense survivin was without effect on apoptosis induced by TRAIL or TRAIL plus Hemin. Although ectopic survivin marginally reduced baseline apoptosis, it had no significant effect on apoptosis induced by TRAIL plus Hemin. The same pattern was found using the combination of STI571 and TRAIL (data not shown). This suggests that the apoptosis-enhancing effects of survivin disruption are limited to apoptosis induced via the mitochondrial pathway.

Survivin disruption in K562 cells enhances Bcr-abl cleavage. Because caspase-dependent Bcr-abl cleavage was described in K562 cells (31) and we reported that survivin blocks caspase activity and caspase-mediated cleavage of Mdm2 (15), we hypothesized that enhanced Bcr-abl cleavage resulting from caspase inhibition may represent the mechanism whereby survivin disruption enhances STI571-induced apoptosis. After STI571 treatment, elevated total and phosphorylated Bcr-abl were observed in K562 cells overexpressing survivin (Fig. 4A), whereas overexpression of T34A or antisense survivin reduced their levels. Identical results were observed for c-abl and phospho-c-abl. Analysis of Bcr-abl cleavage products indicated that reduced Bcr-abl protein on survivin disruption was associated with significantly higher levels of Bcr-abl cleavage products. In contrast, the pan-caspase inhibitor z-VAD-fmk blocked Bcr-abl cleavage in STI571-treated K562 cells expressing T34A or antisense survivin (Fig. 4B). This suggests that the enhanced sensitization to STI571-induced apoptosis observed on disruption of survivin expression/function likely results from enhanced caspase-mediated cleavage of Bcr-abl protein.



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  		Figure 4. Enhanced apoptosis by survivin disruption accompanied with caspase-dependent Bcr-abl cleavage. K562 cells transfected with control vector, WT, T34A, or antisense survivin were cultured with 0.5 µmol/L STI571 for 48 hours. A, whole-cell lysates were subjected to Western blot analysis for Bcr-abl, survivin, tyrosine-phospho-Bcr-abl, c-abl, tyrosine-phospho-c-abl, and ß-actin protein. Cells were cultured for 48 hours in the presence of 0.5 µmol/L STI571 and Bcr-abl and c-abl cleavage products determined using anti–c-abl antibody. Data are from one of three experiments with similar results. B, K562 cells transfected with T34A, or antisense survivin were cultured for 48 hours in the presence of 0.5 µmol/L or STI571 with and without the pan-caspase inhibitor z-VAD-fmk (30 µmol/L) and Bcr-abl and c-abl cleavage products determined. Data are representative of three experiments.

 
Survivin disruption enhances cytochrome c release and caspase-9 activity in STI571-treated K562 cells. Bcr-abl exerts its antiapoptotic effect by blocking mitochondrial release of cytochrome c (6, 7). STI571 induces mitochondrial damage and apoptosis in K562 cells (33). Moreover, survivin disruption results in enhanced caspase-9 activity and sensitizes HeLa cells to apoptosis and loss of {Delta}{Psi}m (34). We therefore investigated whether enhanced apoptosis induced by STI571 and survivin disruption was mediated through an effect on {Delta}{Psi}m. Following treatment with 0.5 µmol/L STI571 for 24 hours, K562 cells overexpressing survivin had lower cytosolic cytochrome c levels and higher mitochondrial cytochrome c compared with control cells (Fig. 5A). Survivin disruption resulted in elevated cytosolic cytochrome c and reduced levels of mitochondrial cytochrome c following treatment with STI571, suggesting that blocking survivin expression/function enhances disruption of {Delta}{Psi}m by STI571. Direct measurement of caspase-9 activity in STI571-treated K562 cells transfected with T34A, C84A, or antisense survivin confirmed that survivin disruption was associated with enhanced caspase-9 activity (Fig. 5B).



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  		Figure 5. Ectopic expression of survivin inhibits cytochrome c release and caspase-9 activity in Bcr-abl Pos K562 cells after STI571 treatment. A, K562 cells transfected with control vector, WT survivin, and T34A survivin were cultured with 0.5 µmol/L STI571 for 48 hours and cytosolic and mitochondrial protein isolated and subjected to Western blot analysis for survivin and cytochrome c. Gel staining was used as loading control. B, caspase-9 activity in cytoplasmic proteins from A was measured as described in Materials and Methods. Columns, mean absorbance at 405 nm from three independent experiments; bars, SE. *, P < 0.005, compared with the vector control.

 
We directly measured the effects of survivin modulation on {Delta}{Psi}m in STI571-treated K562 and Mo7e-bcr-abl cells using the cationic dyes TMRM (21) and JC-1 (22). In both K562 cells (Fig. 6A) and Bcr-abl–transduced Mo7e cells (Fig. 6B), ectopic survivin protected cells from mitochondrial {Delta}{Psi}m disruption induced by STI571 treatment for 24 hours, whereas survivin disruption enhanced {Delta}{Psi}m disruption.



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  		Figure 6. Survivin disruption promotes mitochondrial membrane potential disruption in Bcr-abl–transduced Mo7e and Bcr-ablPos K562 cells after STI571 treatment. Bcr-ablPos Mo7e cells (A) or K562 cells (B) were transfected with WT survivin, antisense survivin, or control vector and cultured with 0.5 µmol/L STI571 for 24 hours, and {Delta}{Psi}m was analyzed by flow cytometry using TMRM. C, K562 cells were transfected with 20-mer antisense survivin oligonucleotide (ODN) or control oligonucleotide, and mitochondrial depolarization was determined by JC-1 expression with or without treatment with 0.5 µmol/L STI571 for 24 hours. C, inset, survivin mRNA expression following oligonucleotide transfection. Data are from two experiments with similar results.

 
Antisense survivin oligonucleotide enhances apoptosis. To model a potential anti-survivin therapeutic strategy, we used a 20-mer survivin antisense oligonucleotide (survivin-AS ODN; ref. 17) to disrupt survivin mRNA and evaluated its effect on {Delta}{Psi}m in STI571-treated K562 cells (Fig. 6C). Transfection of K562 cells with survivin-AS ODN enhanced disruption of {Delta}{Psi}m by STI571 compared with cells transfected with nonsense oligonucleotide, although the survivin-AS ODN significantly reduced, but did not completely block, survivin mRNA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Constitutive activation of the Bcr-abl tyrosine kinase contributes to leukemic transformation and confers survival advantages to leukemic stem cells (3). Ectopic expression of Bcr-abl renders cells resistant to apoptosis induced by diverse stimuli, including most cytotoxic drugs (4). Although the downstream targets of Bcr-abl responsible for apoptosis resistance are not known with certainty, multiple signaling/survival pathways have been implicated (7, 23). Results reported herein now link Bcr-abl to the IAPs survivin and ILP-2.

Elevated survivin mRNA and protein are detected in Mo7e and BaF3 cells transduced with Bcr-abl. Moreover, siRNA knockdown of Bcr-abl reduces survivin protein. Although survivin is mainly expressed in G2-M phase (25), survivin up-regulation was not a result of cell cycle distribution; in fact, a lower proportion of Mo7e-bcr-abl cells were in G2-M phase, as reported for Bcr-abl–transduced HL-60 cells (35). In Mo7e-bcr-abl cells, enhanced survivin promoter activity was observed, suggesting an effect of Bcr-abl on survivin transcription. Enhanced survivin promoter activity was not observed when a minimal –71 to +366survivin promoter was expressed in Bcr-abl–transduced cells, indicating that the effects of Bcr-abl were manifested on the –1,824 nt upstream region. Because Bcr-abl induces transcription of heat shock protein 70 through binding to a "GATA" response element [5'(A/T) GATA (A/G)] (24), we specifically mutated one of the GATA binding sites in the survivin promoter and deleted specific promoter segments containing additional putative GATA binding sites (deletions WT{Delta}2-WT{Delta}6). Only one deletion mutant {Delta}–1,194 to –1,078WT{Delta}3 showed significant reduction (77%) in luciferase activity in Mo7e-bcr-abl cells. This construct also showed reduced activity in parental Mo7e cells, but to a lesser degree (Fig. 2B), suggesting that the effects observed using the WT{Delta}3 deletion are due to the inability of endogenous transcription factor(s) present in these cells to bind and regulate survivin. Because the reduction in luciferase activity was significantly greater in Mo7e-bcr-abl cells, the Bcr-abl–mediated effect likely results from the modulation (i.e., increase in activity, nuclear translocation, or level of transcription factors that bind in this region). TRANSFAC analysis also identified putative binding sites for lymphoid enhancer-binding factor 1 (LEF1), activator protein 1 (AP1), E2F, and SP1 along with GATA and the nuclear receptors glucocorticoid receptor, retinoid X receptor, and vitamin D receptor. The small decrease in activity observed with {Delta}–1,194 to –1,078WT{Delta}3 in control Mo7e cells may be due to deletion of binding sites required for basal transcription. In this regard, the transcription factor SP1 has been shown to regulate survivin expression (36). It is unlikely that Bcr-abl regulation of survivin is mediated through GATA, LEF1, SP1, or nuclear receptors because a decreased luciferase activity was not observed using other mutation/deletion constructs that also contained these binding sites. It is possible that Bcr-abl may activate the survivin promoter through AP1, E2F, or other unknown transcription factor binding sites in this region. Both AP1 and E2F have been shown to be regulated by Bcr-abl (37, 38). It is also worth noting that expression and activity of AP1 are growth factor regulated in Mo7e cells (39). Similarly, E2F is regulated by growth factors (40). Because Mo7e cells are maintained in GM-CSF, it is likely that these transcription factors are present in both control- and Bcr-abl–transduced cells, although in Bcr-abl–transduced cells these transcription factors might be present at a higher level or may be more active. Although Bcr-abl can promote Mdm2 translation through an effect on La antigen without transcriptional activation of Mdm2 mRNA (41), our results show that Bcr-abl transcriptionally up-regulates survivin mRNA, resulting in elevated survivin protein. We also found that Bcr-abl does not affect survivin splicing because survivin-{Delta}Ex3 and survivin-2B were up-regulated to the same extent as WT survivin, suggesting that Bcr-abl exerts its effect on survivin at the transcriptional level and not posttranscriptionally. However, it is possible that Bcr-abl might regulate survivin posttranscriptionally by increasing the half-life of survivin mRNA.

STI571 (Gleevec) produces clinical responses in most CML patients (42); however, the development of drug resistance, either by increased expression of Bcr-abl through gene amplification or development of mutations in the Bcr-abl catalytic domain that interfere with STI571 binding, limits efficacy (26) and points to the need for additional treatment strategies. Disruption of {Delta}{Psi}m and cytochrome c release by survivin disruption indicates that survivin is involved in the blockade of mitochondrial injury and caspase activation conferred by Bcr-abl, and that survivin inhibition of caspase activation represents a therapeutic target downstream of Bcr-abl. One approach to overcome resistance would be to decrease Bcr-abl oncoprotein level and activity. Our results in Mo7e-bcr-abl and K562 cells show that survivin disruption enhances caspase-9 activation and the sequela of reduced total and tyrosine-phosphorylated Bcr-abl. Conversely, survivin overexpression enhances Bcr-abl expression, suggesting the presence of a positive growth-promoting feedback loop between Bcr-abl and survivin. Survivin disruption by T34A survivin or survivin antisense oligonucleotides enhances apoptosis induced by STI571. It will be interesting to evaluate STI571-resistant primary CML cells to determine if survivin disruption can resensitize cells to STI571-induced apoptosis.

In addition to survivin, ILP-2 mRNA and protein were up-regulated in Bcr-abl–transduced Mo7e cells. The fact that expression of other IAPs was unaffected suggests that ILP-2 up-regulation is specific. ILP-2 is a novel IAP with restricted specificity for caspase-9 and, like survivin, contains only a single BIR domain. ILP-2 gene expression has been reported only in normal human testes and a lymphoid B-cell line (43). Our previous analysis of IAP mRNAs in hematopoietic cells (18) predated the identification of ILP-2 and it remains to be determined if ILP-2 is expressed and regulated in normal and leukemia primary stem and progenitor cells, and whether it plays a role in cell cycle progression and apoptosis similar to survivin. If ILP-2 is overexpressed in Bcr-ablPos CML cells, gene disruption may enhance sensitization of cells to STI571 or chemotherapy.

In cells with high Bcr-abl levels, key apoptotic events such as caspase activation and mitochondrial depolarization do not occur in response to cytotoxic drugs, indicating that apoptosis is inhibited either at or above the mitochondrial level (44). STI571 induces mitochondria-dependent apoptosis in K562 cells (31), and survivin blocks caspases 3, 7, and 9 downstream of the mitochondria. K562 cells are resistant to TNF{alpha}, Fas ligand/CD95 ligand, and TRAIL, which typically initiate apoptosis through formation of the death-inducing signaling complex and activation of procaspase-8, which subsequently activates caspase-3 (30, 31). Because survivin partially inhibits apoptosis induced by Fas (32), likely through caspase-3 blockade, and TRAIL induces apoptosis even in Bcl-2– and Bcl-xL–overexpressing tumor cells that are protected against mitochondria-targeting cytotoxic agents (45), we had hoped that survivin disruption would sensitize Bcr-ablPos cells to agents that activate the extrinsic pathway. However, ectopic survivin did not protect K562 cells from apoptosis induced by TRAIL plus Hemin, and survivin disruption did not sensitize cells to apoptosis induced by death receptor activation. The inability of ectopic survivin to protect K562 cells from TRAIL plus Hemin–induced apoptosis may be due to already high levels of endogenous survivin or altered cell death receptor pathways. However, the fact that survivin disruption did not facilitate or sensitize cells to TRAIL-induced apoptosis was unexpected because both extrinsic and intrinsic pathways should be activated. Recent reports, however, have shown that one of the contributing factors to leukemic cell (K562) resistance to TRAIL may be aberrant caspase-8 activity (46, 47). In addition, survivin itself has been shown to be up-regulated in TRAIL-treated cells (46). Thus, the lack of cooperation between survivin disruption and TRAIL in inducing apoptosis might be due to up-regulation of survivin by TRAIL and lack of caspase-8 activation or a balance between stimulation and blockade of apoptosis. It is also possible, as in the case of Bcl-2, which protects prostate cancers but not lymphoid tumors from TRAIL-mediated apoptosis (48, 49), that survivin disruption overcomes TRAIL resistance only in solid tumors (50, 51). This suggests that anti-survivin therapies may only have utility in combination with agents such as CDDP and Ara-C, which induce apoptosis via the intrinsic pathway (28, 29). Although this potentially rules out the combination of TRAIL and survivin disruption in treatment of CML, TRAIL is more potent in selectively killing solid cancer cells (52), and in the absence of toxicity to hematopoietic progenitors, use of this combination may have utility in nonhematologic cancers.

Full-length survivin transcripts are highly expressed in cancer cells and detected at low levels in many normal adult tissues (23, 53). The alternatively spliced survivin variants, {Delta}Ex3, which retains antiapoptotic activity, and 2B, which has reduced antiapoptotic activity, at least in HepG2 cells (54), are expressed in most cancer cells, but are not readily detected in normal cells (53). We detected all three survivin transcripts in parental Mo7e cells and in normal CD34+ cells,1 and consistent with published reports, full-length survivin is the dominant transcript. In keeping with an effect of Bcr-abl on survivin promoter activity, all three survivin transcripts were elevated in Bcr-abl–transduced Mo7e cells. In contrast, differential regulation of survivin-2B by p53 was recently described in an ALL cell line, which sensitized cells to doxorubicin (55). Because survivin expression may be involved in the progression of Bcr-ablPos CML to acute leukemia (56), it will be of interest to determine the abundance of each of the survivin splice variants in primary CML cells and identify whether their level/distribution changes with respect to phase of disease and/or response to therapy, particularly STI571.

In summary, we show that the Bcr-abl oncogene elevates survivin, which represents a new downstream signal pathway that may be helpful in understanding the pathogenesis and pathophysiology of CML. Targeted survivin disruption may sensitize Bcr-ablPos CML cells to apoptosis induced by STI571 and may have therapeutic potential. In addition, the finding that Bcr-abl also up-regulates ILP-2, at least in leukemic Mo7e cells, warrants further investigation to determine if anti–ILP-2 targeted therapy may also have utility in Bcr-ablPos CML.


   Acknowledgments
 
Grant support: NIH grants HL079654 and HL69669 (L.M. Pelus).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. H. Scott Boswell (Indiana University, Indianapolis, IN) for the Bcr-abl–transduced Mo7e cells, Dr. M.W. Deininger (Oregon Health and Science University, Portland, OR) for the Bcr-abl–transduced BaF3 cells, and Dr. Suxin Liu (Schering-Plough Research Institute, Kenilworth, NJ) for the full-length survivin promoter plasmid.


   Footnotes
 
1 S. Fukuda and L.M. Pelus, unpublished data. Back

Received 1/28/05. Revised 6/22/05. Accepted 7/ 1/05.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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