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[Cancer Research 65, 210-218, January 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

cIAP1 Localizes to the Nuclear Compartment and Modulates the Cell Cycle

Temesgen Samuel, Kazuya Okada, Marc Hyer, Kate Welsh, Juan M. Zapata and John C. Reed

Burnham Institute, La Jolla, California

Requests for reprints: John C. Reed, Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: 858-646-3140; Fax: 858-646-3194. E-mail: reedoffice{at}burnham.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We explored the location and function of the human cIAP1 protein, a member of the inhibitor of apoptosis protein (IAP) family. Unlike family member X-linked IAP (XIAP), which was predominantly cytoplasmic, the cIAP1 protein localized almost exclusively to nuclei in cells, as determined by immunofluorescence microscopy and subcellular fractionation methods. Interestingly, apoptotic stimuli induced nuclear export of cIAP1, which was blocked by a chemical caspase inhibitor. In dividing cells, cIAP1 was released into the cytosol early in mitosis, then reaccumulated in nuclei in late anaphase and in telophase, with the exception of a pool of cIAP1 that associated with the midbody. Survivin, another IAP family member, and cIAP1 were both localized on midbody microtubules at telophase, and also interacted with each other during mitosis. Cells stably overexpressing cIAP1 accumulated in G2-M phase and grew slower than control-transfected cells. These cIAP1-overexpressing cells also exhibited cytokinesis defects over 10 times more often than control cells and displayed a mitotic checkpoint abnormality with production of polyploid cells when exposed to microtubule-targeting drugs nocodazole and paclitaxel (Taxol). Our findings demonstrate a role for overexpressed cIAP1 in genetic instability, possibly by interfering with mitotic functions of Survivin. These findings may have important implications for cancers in which cIAP1 overexpression occurs.

Key Words: IAP • subcellular localization • cytokinesis defect • genetic instability • cancer


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inhibitor of apoptosis proteins (IAP) are a family of proteins containing one or more zinc-binding folds called baculoviral IAP repeat (Bir) domains. IAPs are involved in various cellular functions, including regulation of apoptosis, cell cycle, and intracellular signal transduction (1, 2). IAP family proteins have been identified in viruses, yeast, insects, worms, and vertebrates. The human genome contains eight IAP-encoding genes (3).

Several IAP family members function predominantly in regulating apoptosis. For example, X-linked IAP (XIAP) is a potent and direct inhibitor of caspases 3, 7, and 9 (4–6) and is involved in protection from Fas-mediated apoptosis (7). In addition to direct caspase inhibition, recent studies have revealed additional roles for XIAP in several pathways directly or indirectly connected to apoptosis. Accordingly, XIAP has been found to modulate the transforming growth factor ß signaling through Alk3 and Alk5 (8) and c-jun-NH2-kinase signaling through TAK1 and ILPIP (9–12). Moreover, a TAK1-dependent mechanism for the nuclear factor-{kappa}B activation by XIAP has also been described (13). Other mammalian IAP family members, cIAP1 and cIAP2, have also been shown to directly inhibit caspase activity, albeit weakly compared to XIAP (14). Additionally, cIAP1 and cIAP2 modulate death receptor signaling by interacting with the TRAFs in tumor necrosis factor (TNF)-receptor complexes (15, 16).

Survivin, the smallest IAP member with a single Bir domain, regulates both the cell cycle and apoptosis (17). Whereas the cell cycle function of Survivin is dependent on its association with the chromosomal passenger proteins (INCENP, Aurora B, and Borealin) at the kinetochore (18–23), the apoptosis functions have been related to its interaction with cofactors such as HBXIP, which facilitates interactions with procaspase-9 (24), and to interactions with other IAPs that directly bind caspases (25). Survivin and procaspase-9 reportedly colocalize in mitotic cells (26), suggesting a role for Survivin in controlling a cell cycle checkpoint that results in cell suicide if improperly executed (27–29).

Despite the overall sequence and structural similarity among the IAP family members, the subcellular distribution of IAPs seems extremely variable. For example, Survivin in normal cells is predominantly a nuclear protein, the expression of which is cell cycle dependent, peaking at G2-M (30). On the other hand, most tumor cells show nuclear and cytoplasmic Survivin distribution, which is cell cycle independent (31). Another member of the IAP family, XIAP, is expressed mostly in the cytoplasm, but in the presence of the antagonist protein, XAF1, is sequestered in nuclei (32). Expression of cIAP1 has been suggested to be cytoplasmic, nuclear, or both (33–36). Surprisingly, cIAP2 expression has been detected also in mitochondrial fractions (33). The mechanisms regulating the distribution of IAPs into various subcellular compartments and the correlation of their subcellular location with protein function require further clarification.

In this study, we examined the intracellular distribution of cIAP1, the contribution of the Bir domains to localization of cIAP1, and explored whether certain stimuli alter cIAP1 localization within cells. Our findings indicate that cIAP1 is predominantly a nuclear protein, and its nuclear localization is mediated by the Bir domains. We also report that overexpression of cIAP1 induces aberrant cell division and accumulation of polyploid cells through defective cytokinesis. Moreover, a pool of cIAP1 concentrates at the midbody of telophase cells, suggesting a potential role for cIAP1 in the cell cycle. We also present evidence that cIAP1 interacts with Survivin, possibly linking cIAP1 overexpression to the cytokinesis phenotype produced by cIAP1 overexpression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and Reagents. Antibodies for immunoblotting and fluorescent microscopy were obtained from the following sources: cIAP1 and cIAP2 (R&D systems, Minneapolis, MN); cIAP1 monoclonal antibody, XIAP, and poly(ADP-ribose) polymerase (PARP, BD PharMingen, San Diego, CA); myc clone 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA); p53 Ab-6 (Calbiochem, SanDiego, CA); Flag-M2 and actin (Sigma, St. Louis, MO). The rabbit anti-Survivin antibody has been described (37). Other reagents purchased were etoposide (Calbiochem), staurosporine and nocodazole (Sigma), TNF-related apoptosis-inducing ligand (TRAIL) and TNF-{alpha} (Biomol, Plymouth Meeting, PA), and 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF, Roche Diagnostics, Mannheim, Germany). Alexa Fluor–conjugated secondary antibodies were purchased from Molecular Probes (Eugene, OR).

Plasmids. Generation of the full-length cIAP1 was described previously (14). Plasmids encoding individual fragments of cIAP1 corresponding to Bir1, Bir2, Bir3, and Bir3-RING were generated by PCR and cloned into pcDNA3, in-frame with an NH2 terminal myc epitope tag.

Cell Culture, Transfections, and Treatments. HeLa and HCT116 cells were grown in DMEM (Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal bovine serum, glutamine, and penicillin/streptomycin. Du145 cells were grown in RPMI medium (Irvine Scientific) supplemented as above. Transfections were done using Lipofectamine 2000 (or Lipofectamine Plus reagents) and OptiMem reduced serum medium (Invitrogen, Carlsbad, CA). Lipofectamine transfection mix was removed after 4 hours. Independent clones of Du145-cIAP1 stable transfectants were selected with600 µg/mL G418. For some experiments, transfected or untransfected HeLa cells were treated with TRAIL (200 ng/mL), TNF (20 ng/mL), staurosporine (0.7µmol/L), or UV irradiation (0.12 J/cm2) from UV Stratalinker (Stratagene, La Jolla, CA). G2 synchronization of HeLa cells was achieved by treatment with 40 ng/mL nocodazole for 14 hours. Cells were released from the block by washing twice with complete medium. For subsequent experiments, cells were harvested 90 minutes after release from the block.

Subcellular Fractionation. Cells were harvested in an enzyme-free cell dissociation solution (specialty medium, Cell and Molecular Technologies, Inc., Phillipsburg, NJ), washed once in cold PBS, and gently resuspended in ~1.5 pellet volume of ice-cold hypotonic lysis buffer [300 mmol/L sucrose, 10 mmol/L KCl, 10 mmol/L HEPES (pH 7.4), 1.5 mmol/L MgCl2, 0.1 mmol/L EGTA, 0.5 mmol/L DTT, 0.3% NP40, and "Complete Mini" protease inhibitor mixture (Roche Diagnostics)]. The cell suspension was incubated on ice for 10 minutes. Complete lysis was ensured by microscopy analysis of small aliquots of the suspension, allowing more time for lysis when necessary. The lysate was centrifuged at 800 x g for 2 minutes at 4°C, and the supernatant was carefully collected, and stored as cytosolic extract. The pellet was resuspended in 0.5 mL of hypotonic lysis buffer and washed in lysis solution, recovering nuclei by centrifugation at 800 x g for 4 minutes at 4°C). The supernatant was removed and nuclear pellet was resuspended in modified radioimmunoprecipitation assay buffer [150 mmol/L KCl, 20 mmol/L HEPES (pH 7.4), 1% Triton X-100, 0.5% deoxycholate, 1% SDS, 5 mmol/L EDTA, and protease inhibitors]. Fractions were normalized for cell number and analyzed by SDS-PAGE and immunoblotting.

Immunoprecipitation. For immunoprecipitation experiments, loosely adherent mitotic cells were harvested from culture dishes by forceful washing with PBS and centrifuged at 290 x g. The cell pellets were resuspended in 2x pellet volume of coimmunoprecipitation lysis buffer [20mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.2% NP40, 10% glycerol, and protease inhibitor mixture] containing 1% SDS and boiled for 5 minutes at 95°C. Any insoluble material was removed by centrifugation at 16,000 x g for 20 minutes at 4°C. The supernatant was transferred to a new tube and the final volume was adjusted to 10x pellet volume by adding coimmunoprecipitation lysis buffer containing no SDS. Immunoprecipitation was carried out using Protein-A beads and rabbit anti-Survivin antibody. After an overnight incubation, beads were washed thrice with coimmunoprecipitation lysis buffer, and bound proteins were eluted and analyzed by immunoblotting.

In vitro Protein Interaction Assays. Glutathione S-transferase (GST) fusion proteins were produced from pGEX4T-1 plasmid in XL1-blue cells (Stratagene) and affinity purified using glutathione-Sepharose. In vitro translated proteins were produced using the TNT Quick Coupled Transcription System (Promega, Madison, WI). A total of 2.5 µL of 35S-labeled in vitro translated protein mix was incubated with purified proteins (0.5-1.0 µg) immobilized on 20 µL of glutathione-Sepharose beads in 0.1 mL of binding buffer [50 mmol/L Tris-HCl (pH 7.5), 5 mmol/L MgCl2, 10% glycerol, 0.5 mg/mL bovine serum albumin and 5 mmol/L 2-mercaptoethanol] at 4°C for 60 minutes. The beads were washed thrice with 1.0 mL binding buffer followed by boiling in 25 µL of SDS sample buffer, and proteins were analyzed by SDS-PAGE (12%). Use of equivalent amounts of intact purified proteins and successful in vitro translation of all proteins was confirmed by SDS-PAGE analysis using Coomassie staining or autoradiography, respectively (data not shown).

Immunofluorescence/Confocal Microscopy. For indirect immunofluorescence microscopy, cells were grown on 22-mm-diameter coverslips. Cells transfected on coverslips were examined 24 hours after transfection. Cells were fixed on coverslips using freshly made 3.7% formaldehyde (containing 1-1.5% methanol) in PBS (pH 7.4) for 15 minutes, permeabilized with 0.1% Triton X-100 in PBS for 10 minutes, and preblocked with 2% bovine serum albumin in PBS for 1 hour. Primary antibodies were diluted in 1% bovine serum albumin and PBS and incubated for 1 hour. After washing, cells were incubated with secondary antibodies conjugated to Alexa Fluor 488 (green), or Alexa Fluor 594 (red) dyes (Molecular Probes). Actin cytoskeleton was stained with phalloidin conjugated to Alexa Fluor dyes (Molecular Probes). Finally, coverslips were mounted on microscope slides using Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (DAPI). Cells were visualized by laser-scanning confocal microscopy (Bio-Rad, Hercules, CA) or by using a Leica DIMRE2 microscope equipped with Z-focus motor and SimplePCI (Compix, Inc., Tualatin, OR) image capturing and analysis software (version 5.1.0.0110).

Flow Cytometry. Cells were harvested by trypsinization and washed once with PBS. Washed cells were resuspended in 0.3 mL PBS (pH 7.4), and fixed by addition of 100% ethanol while vortexing. Fixation proceeded for at least overnight at –20°C. Fixed cells were centrifuged, resuspended in propidium iodide solution (40 µg/mL propidium iodide, 320 µg/mL RNase-A in PBS without calcium and magnesium), and incubated for 15 minutes at 37°C. Stained cells were passed through nylon-mesh sieve to remove cell clumps and analyzed by a FACScan flow cytometer and CellQuest analysis software (Becton Dickinson, San Jose, CA).

Carboxyfluorescein Diacetate Succinimidyl Ester Dilution Assay. Carboxyfluoresce in diacetate succinimidyl ester (CFDA SE, also commonly called CFSE) was obtained from Molecular Probes. Du145 cells were plated at a density of 105 cells per well in six-well dishes. At 16 hours after seeding, cells were labeled with 10 µmol/L CFSE in PBS for 15 minutes at 37°C according to the manufacturer's recommendations. After 15 minutes, the labeling solution was replaced with fresh, prewarmed medium. Cells were collected by trypsinization at various times and processed for flow cytometry as above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
cIAP1 Localizes to Nuclei. To establish the subcellular distribution of cIAP1, we did cell fractionation experiments using HeLa cells. Examination of the fractions for cIAP1 and other endogenous apoptosis-related proteins showed that cIAP1 and the control protein, PARP, localized to the nuclear fractions, whereas XIAP, Smac, and pro-caspase 3 localized to cytosolic fractions (Fig.1A). To complement the fractionation experiments, we did immunofluorescence staining for endogenous cIAP1, cIAP2, XIAP, Survivin, and p53 in HeLa cells, using the DNA-binding fluorochrome DAPI, phalloidin, or anti-tubulin antibody as counterstains with indirect immunofluorescence detection. Fluorescence microscopy confirmed the results from subcellular fractionation studies, indicating that cIAP1 is a primarily nuclear protein (Fig. 1B and C). As shown in Fig. 1A and C, cIAP2 localized to both cytoplasmic and nuclear compartments, whereas XIAP was mostly cytoplasmic. Survivin and p53 localized to the nuclear compartment.



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  		Figure 1. Comparison of subcellular localization of IAPs. A, cytosolic (C), nuclear (N), and whole-cell lysates prepared from HeLa cells were analyzed by SDS-PAGE and immunoblotting using antibodies specific for cIAP1, XIAP, Smac, PARP, Caspase-3 and cIAP2. *, nonspecific band. B, nuclear localization of cIAP1 was confirmed by immunofluorescence staining of HeLa cells for endogenous cIAP1. Cells were stained for cIAP1 (green) and counterstained with DAPI as nuclear marker (top) or tubulin as cytoplasmic marker (bottom). C, immunofluorescent staining for endogenous IAP proteins (cIAP1, cIAP2, XIAP, Survivin) and p53. HeLa cells were stained with primary antibodies to the indicated proteins (red), and counterstained (green) with anti-tubulin antibodies (in conjunction with rabbit primary antibodies to cIAP1, cIAP2, Survivin) or phalloidin-Alexa Fluor 488 (with mouse primary antibodies to XIAP and p53).

 
The Bir Domains of cIAP1 Mediate Nuclear Localization. We analyzed the domains in cIAP1 responsible for its nuclear targeting. Direct examination of the cIAP1 sequence revealed no amino acid sequence motifs meeting criteria for potential nuclear localization signal sequences. Therefore, to determine which domains of cIAP1 confer nuclear localization to the protein, we expressed fragments of the cIAP1 protein, with NH2-terminal myc tags, representing the Bir1, Bir2, Bir3, and Bir3-RING domains, as well as the full-length protein. Immunofluorescence microscopy was then used in conjunction with an antibody directed against the myc tag to localize these cIAP1 fragments in transfected HeLa cells (Fig. 2).



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  		Figure 2. Ectopically expressed cIAP1 localizes to the nucleus. HeLa cells were transfected with myc-tagged Bir1, Bir2, Bir3-RING, or full-length cIAP1 constructs and the subcellular localization of the expressed proteins was examined by immunofluorescence microscopy 12 hours (A) or 24 hours (B and C) after transfection. Immunostaining was done using primary anti-myc antibody followed by Alexa Fluor 488–conjugated anti-mouse secondary antibody. Nuclei were counterstained with DAPI. D, HCT116 cells were transfected with a plasmid encoding Flag-tagged full-length cIAP1. Cells were harvested after 24 hours, and cytosolic and nuclear fractions were analyzed. Overexpressed cIAP1 was detected by immunoblotting of the fractions with anti-Flag antibody. Immunoblotting for Smac and PARP proteins served as control for proper cytosolic-nuclear protein segregation.

 
At 12 hours post transfection, the expressed proteins were mostly cytoplasmic (Fig. 2A). However, after 24 hours, all Bir domains of cIAP1 localized to nuclei (Fig. 2B and data not shown). We also observed that expression of the Bir3-RING fragment of cIAP1 resulted in diffuse staining of the expressed protein, where the Bir3-RING protein was detected in both the cytoplasmic and nuclear compartments. The full-length protein, however, was entirely nuclear, indicating the probable dominance of the Bir domains on the overall distribution of cIAP1 (Fig. 2C). To ensure that the nuclear localization of the ectopically expressed proteins was not influenced by the myc tag, we also expressed Flag-tagged full-length cIAP1 in HCT116 cells and analyzed the localization of the expressed protein in cytosolic-nuclear fractions by immunoblotting. As shown in Fig. 2D, the Flag-tagged cIAP1 was also exclusively detected in the nuclear fractions. The purity of the fractions was controlled by immunoblotting for Smac and PARP proteins, which were localized to the cytosolic and nuclear fractions, respectively. These results indicate that both endogenous and ectopically expressed cIAP1 target to nuclei.

Inducible Relocalization of cIAP1. Because most of the known functions of cIAP1 require cIAP1 to be in the cytoplasm, we examined whether the subcellular location of cIAP1 changes in response to various apoptotic stimuli. To this end, we treated HeLa cells with various apoptotic agents that trigger cell death through either the death receptor-mediated pathway (e.g., TRAIL) or the mitochondria-dependent pathway (e.g.,DNA-damaging agents, such as UV irradiation, or the broad-spectrum kinase inhibitor, staurosporine. The location of cIAP1 was then examined by subcellular fractionation (Fig. 3A and B).



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  		Figure 3. Nuclear-cytoplasmic redistribution of cIAP1. A, Cytoplasmic and nuclear fractions prepared from HeLa cells left untreated or treated with UV irradiation (0.12 J/cm2), TRAIL (200 ng/mL), or staurosporine (STS, 0.7 µmol/L) were analyzed by immunoblotting for cIAP1, Smac, and PARP. Cells were harvested 2 hours (STS and TRAIL) or 3 hours (UV) after treatment. B, caspase inhibition blocks cIAP1 relocalization. HeLa cells were left untreated (top) or treated with TRAIL to induce cIAP1 relocalization (bottom) with or without z-VAD-fmk or the serine protease inhibitor AEBSF. Cytoplasmic and nuclear fractions were prepared and analyzed for the distribution of cIAP1 (top), Survivin, PARP, and Smac by immunoblotting. C, HeLa cells transfected with plasmid encoding myc-cIAP1 were left untreated or treated with TNF-{alpha} (20 ng/mL) 24 hours after transfection, and localization of the expressed protein was determined by indirect immunofluorescence staining using anti-myc antibody (top). Location of endogenous cIAP1 protein in HeLa cells that were untreated or were exposed to TNF-{alpha} (20 ng/mL) plus cycloheximide (CHX, 10 µg/mL), was determined by indirect immunofluorescence staining (bottom). Images from representative cells are presented. D, percentage of cells with cytosolic localization of cIAP1 was determined following treatment with TNF-{alpha} and CHX as above. Data indicate percentage of cells showing cytoplasmic relocalization of myc-cIAP1 or endogenous cIAP1 proteins, based on counting of ≥100 cells from at least four microscopic fields.

 
We found that several agents that induced either the extrinsic or intrinsic apoptotic pathways promoted the redistribution of cIAP1 from the nuclear compartment to the cytoplasmic compartment, including TNF, TRAIL, staurosporine, and UV irradiation (Fig. 3A-C). Under the same conditions, PARP was always detected in the nuclear fractions, whereas Smac remained cytoplasmic. The process of cIAP1 redistribution correlated with caspase activation, as evidenced by PARP cleavage (Fig. 3A and B). Therefore, to examine if inhibition of caspases would block the redistribution of cIAP1, HeLa cells were treated with TRAIL in the absence or presence of the broad-spectrum caspase inhibitor z-VAD-fmk or the serine protease inhibitor AEBSF. Then, cytosolic and nuclear fractions were analyzed for cIAP1, Survivin, PARP, and Smac proteins. As shown in Fig. 3B, the nuclear-cytoplasmic redistribution of cIAP1 was suppressed by inhibition of caspases but not by inhibition of serine proteases. Moreover, z-VAD-fmk but not AEBSF inhibited the cleavage of PARP. Neither z-VAD-fmk nor AEBSF affected the subcellular distribution of Survivin, PARP, and Smac in TRAIL-treated HeLa cells.

To corroborate the results from cell fractionation experiments, we undertook immunomicroscopy-based localization of cIAP1 using HeLa cells exposed to TNF. cIAP1-transfected or untransfected cells were treated with TNF-{alpha} with or without cycloheximide. Cells were treated 24 hours after transfection when the overexpressed protein was found predominantly in nuclei. After TNF treatment, the proportion of cells displaying nuclear and cytoplasmic staining for cIAP1 was quantified. As shown in Fig. 3C and D, a significant amount of cIAP1 was found in the cytoplasm after TNF treatment. Interestingly, whereas the subcellular redistribution of exogenously expressed cIAP1 was readily induced by TNF-{alpha} without requirement for cycloheximide (~49 ± 14%), the endogenous cIAP1 protein translocated efficiently only after treatment with the combination of TNF-{alpha} and cycloheximide (~83 ± 7% TNF plus cycloheximide versus 10 ± 4% TNF only).

Localization of cIAP1 Protein in Dividing Cells. To date, Survivin is the only IAP member shown to directly regulate the cell cycle through interaction with chromosomal passenger proteins (reviewed in refs. 19, 38). We determined the location of cIAP1 during the cell cycle, making comparisons with Survivin. We used indirect immunofluorescence microscopy for these experiments, staining replicate cultures of HeLa cells to detect endogenous cIAP1 or Survivin (Fig. 4A). Parallel staining was done because both primary antibodies for immunostaining were raised in rabbits. These antibodies were tested from among multiple commercially available and self-produced reagents and shown to be monospecific for the intended proteins (37).



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  		Figure 4. Assessment of cIAP1 location during cell division. A, HeLa cells grown on coverslips were immunostained for endogenous cIAP1 (left) or Survivin (right), using affinity-purified rabbit primary antibody and Alexa Fluor 488–conjugated anti-rabbit secondary antibody. Representative images are shown for cells at interphase, prophase, metaphase, anaphase, or telophase of the cell cycle, using DAPI as DNA marker. B, higher magnification (63x objective) image of midbody staining of cIAP1. Cells were stained for endogenous cIAP1 (green), whereas counterstaining was done with a monoclonal anti-tubulin antibody (red). Right, parallel staining using a control rabbit antiserum, also counterstained with anti-tubulin antibody (red). The nuclei were counterstained with DAPI (blue). Percentage of telophase HeLa cells that displayed midbody staining with cIAP1-specific and control rabbit antisera (right). C, HeLa cell lysates were prepared from asynchronously growing (Asyn.), nocodazole blocked (G2), or cells released from nocodazole block (Rel.). Survivin (SVV) was immunoprecipitated using rabbit polyclonal antibody and coimmunoprecipitated material was analyzed for the presence of cIAP1 using a mouse monoclonal antibody. Control immunoprecipitation was preformed using a preimmune rabbit serum. Lysates were also immunoblotted for cIAP1 and Survivin proteins (bottom). D, GST (control) or GST fusion proteins containing the Bir1, Bir2, or Bir3 domains of cIAP1 (5 µg) were immobilized on glutathione-Sepharose and incubated with in vitro translated 35S-labeled myc-tagged Survivin or Smac. After 1 hour, beads were washed extensively and analyzed by SDS-PAGE/autoradiography. As a control, 0.1 volume of input 35S-Survivin or Smac protein was loaded directly into the gel. Note that Survivin predominantly binds to Bir2, whereas Smac predominantly binds Bir3 of cIAP1.

 
Immunofluorescence staining of HeLa cells at different phases of the cell cycle revealed that although endogenous cIAP1 is nuclear during interphase, it becomes diffusely distributed throughout the cell from prophase to anaphase, and then recovers during anaphase/telophase, when nuclei reassemble. Interestingly, a pool of cIAP1 also localized to the midbody (Fig. 4A and B), a structure that holds recently replicated cells together until the final stages of cytokinesis split them into two cells. Use of control antibody confirmed the specificity of this midbody staining in telophase cells (Fig. 4B). Western blot examination of cIAP1 protein levels using extracts made from thymidine-synchronized cells revealed no remarkable differences in the steady-state levels of cIAP1 during the cell cycle (data not shown).

Similar to cIAP1, Survivin was found diffusely through nuclei in interphase cells. Unlike cIAP1, however, after nuclear envelop breakdown, Survivin localized to kinetochores of cells in metaphase, then distributed to the midzone microtubules in anaphase. At telophase, Survivin was localized exclusively to the midbody (Fig. 4A), consistent with previous reports (21, 39). Thus, cIAP1 and Survivin are both found associated with the midbody in telophase cells, and are both found diffusely in the nucleus of interphase cells, but were not detectably colocalized during metaphase and anaphase.

Recently, it was reported that XIAP can bind Survivin via a Bir-Bir interaction (25). Given that cIAP1 colocalizes with Survivin during certain cell cycle phases, we questioned whether cIAP1 can associate with Survivin. To this end, coimmunoprecipitation experiments were preformed, using nocodazole-synchronized HeLa cells. A small proportion of cIAP1 was recovered in Survivin immunoprecipitates from late mitotic cells, suggesting these proteins either directly or indirectly associate in cells (Fig. 4C). We also did in vitro protein binding experiments, using GST-tagged fusion proteins produced and purified from bacteria. GST-cIAP1 bound in vitro translated [35S]-Survivin, with Bir2-containing fragments displaying the strongest binding, further suggesting that cIAP1 is capable of interacting with Survivin (Fig.4D).

Cell Growth and Cell Cycle Defects Induced by Ectopic cIAP1. To further study cIAP1 function during the cell cycle, we generated stable transfectants of diploid Du145 human prostate carcinoma cells overexpressing cIAP1. Among a total of nine positive clones obtained (Fig. 5A), two clones (3 and 4) were randomly selected for subsequent studies. Both clones displayed similar phenotypes, although data are presented here only for clone3. Note that the cIAP1-overexpressing clones were examined for only 8 to 10 tissue culture passages because the phenotype became progressively more aberrant, then cIAP1 detection declined with increasing passage number.



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  		Figure 5. Growth characteristics of Du145-cIAP1 cells. A, Du145 human prostate cancer cells were stably transfected with a plasmid encoding a Flag-tagged full-length cIAP1, and 12 independent clones were examined by immunoblotting for expression of Flag-cIAP1 using anti-Flag antibody. The membrane was reprobed with anti-actin antibody to control for protein loading. B, cytokinesis and growth defects in Du145-cIAP1 cells. The periphery of a growing colony was photographed to show a representative example of the cell division defect. Inset, a high-power-magnification view of the impaired separation of two daughter cells. Right, neo-control or cIAP1-transfected cells were seeded at a density of 104 per well in six-well dishes. On subsequent days, cells were detached and counted, and the total number of cells per well was calculated. C, cell proliferation was assessed by CFSE dye dilution assay. Du145-neo or -cIAP1 cells were labeled with CFSE and then returned to culture. At the indicated times, the level of CFSE in labeled cells was measured by flow cytometry on logarithmic scale, and the geometric mean fluorescence was determined (Y axis). Numbers along the curves, percentage of retained CFSE relative to the measured maximum at the beginning of the experiment. D, progressive increase in accumulation of cells with =4n DNA content in cIAP1 cells. Du145-neo control or -cIAP1 cells were harvested at different passage numbers and analyzed by flow cytometry for DNA content by gating on viable cell population. A representative histogram of neo-control cells at passage 8 is provided alongside histograms obtained at the 2nd, 5th, 7th, and 8th passages of Du145-cIAP1 cells. The percentage of cells having =4n DNA content is indicated.

 
We studied the cell growth characteristics of cIAP1 and vector control Du145 cells. In cultures of cIAP1-overexpressing cells, cells at the periphery of expanding colonies often displayed cytokinesis defects (Fig. 5B). Such aberrant cell divisions were rarely seen in cultures of vector-transfected cells (0.7 ± 0.2% versus 8.4 ± 2% in cIAP1 cells).

To measure the rate of population doubling, we plated equivalent numbers of cIAP1 and vector control cells and followed the population growth by counting the number of cells in culture on subsequent days. As shown in Fig. 5B (right), Du145-cIAP1 cells had a substantially lower rate of cell expansion.

Because we saw no increase in cell death to account for the reduced growth rate, we hypothesized that cell division defects were responsible for the apparent slow growth of Du145-cIAP1 cells. To test this hypothesis, we measured growth of Du145-cIAP1 and vector control cells by comparing the rate of decline of incorporated CFSE during cell division (Fig. 5C). Upon uptake by cells, the ester groups of CFSE are cleaved by intracellular esterases, generating intracellular fluorescent products that are unable to vacate cells. As cells with incorporated CFSE divide, the dye fluorescence intensity is proportionally halved, which can be measured by the decrease in mean fluorescence using flow cytometry. As shown in Fig. 5C, the CFSE mean fluorescence in vector control cells steadily decreased over time, whereas cIAP1 cells retained a significant proportion of the initial fluorescence. This result confirmed that cIAP1 Du145 cells had pronounced cell division defects. Taken together, these observations suggest that the slow growth of cells might be associated with defective cell cycle or mitotic exit.

We therefore assessed the cell cycle status of vector control–transfected and cIAP1-transfected cells by DNA content analysis, using flow cytometry. cIAP1-overexpressing cells had significantly higher proportions of cells in the G2-M phase of the cell cycle (4nDNA content), and the DNA content per cell was remarkably polymorphic, suggesting defective cytokinesis, which might have led to nuclear fusion and polyploidy (data not shown, and Figs. 5 and 6). DNA content analysis of Du145-cIAP1 cells suggested that the proportion of cells in the G2-M phase increased with increasing passage, reaching a plateau of 60% to 66% cells with ≥4n DNA content between the 7th and 8th passages (Fig. 5D). Du145-cIAP1 cells also had a reduced percentage of cells in S phase at later passages compared to the vector control–transfected cells (not shown). In contrast, the percentage of cells with ≥4n DNA content did not exceed 31 ± 4% in cultures of vector control Du145 cells. Thus, cIAP1-overexpressing cells may have difficulties in completing mitosis. Moreover, treatment of Du145-neo and -cIAP1 cells with microtubule-targeting agents nocodazole and paclitaxel (Taxol) led to significantly higher proportion of cIAP1 cells entering 8n state compared to vector control cells (Fig. 6), further suggesting defective control of mitotic arrest in Du145-cIAP1 cells.



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  		Figure 6. Defective mitotic arrest in Du145-cIAP1 cells. Du145-neo or -cIAP1 cells were cultured untreated or treated with nocodazole (Nocod., 40 ng/mL) or Taxol (0.2 µmol/L) for 16 hours. Cells were then harvested and analyzed by flow cytometry for DNA content. The histograms show representative DNA content profiles (2n, 4n, or 8n) of untreated (none) and treated (Nocod. and Taxol) Du145-neo and -cIAP1 cells. The proportion of Du145-neo and -cIAP1 cells in the histograms with 2n, 4n, and >4n DNA content is indicated in the table. Representative data from two independent experiments is shown here.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we show that IAPs localize to different subcellular compartments, presenting data on the nuclear localization and subsequent export of cIAP1, as well as the dynamics of intracellular cIAP1 targeting during the cell cycle. We also show that each of the Bir domains of cIAP1 is capable of independently localizing to the nucleus. Previous analysis of the Bir domains of viral IAPs had suggested that Bir domains are capable of binding metal ions and nucleic acids (40). Although there is no evidence that Bir domains can directly bind DNA, our report suggests that the nuclear localization of cIAP1 is a function of its Bir domains and not the RING or CARD domain. While this article was under preparation, two independent reports (36, 41) also showed evidence of cIAP1 targeting to nuclei, and mapped putative Nuclear Export Sequences to the CARD and the region between Bir2 and Bir3. Interestingly, Plenchette et al. (36) further showed that cIAP1 translocates from the nucleus to the Golgi apparatus in cells undergoing differentiation.

Nuclear localization and export of cIAP1 are particularly interesting because cIAP1 has thus far been described to function in regulating the cytosolic activities of caspases and Smac and because it also modulates TNF signaling in extranuclear compartments. We observed that a pool of cIAP1 redistributes into the cytosolic compartment when cells are exposed to certain apoptotic stimuli, including agents that trigger the extrinsic pathway (e.g., TNF, TRAIL) or intrinsic pathway (e.g., UV irradiation, staurosporine). The export of cIAP1 from the nucleus seems to depend on caspase activation, as the broad-spectrum caspase inhibitor z-VAD-fmk blocked the cytoplasmic redistribution of cIAP1. At this stage, we can only hypothesize how caspase activation might promote export of cIAP1. Caspase activity could be required to compromise nuclear membrane integrity, allowing the protein to readily leave the nucleus, or, alternatively, caspases may cleave yet unknown cIAP1-anchoring proteins that normally keep it sequestered in the nucleus. It should also be noted that because others have observed nuclear export of cIAP1 during cell differentiation (36), the possibility exists of both caspase-dependent and-independent mechanisms for triggering export of this protein. However, given that the process of differentiation has been shown to involve moderate caspase activation in some cellular contexts (reviewed in ref. 42), differentiation-associated nuclear export of cIAP1 could also involve caspases.

Because activated caspases cleave nuclear substrates, it is possible that under physiologic conditions cIAP1 regulates apoptotic signaling in the nucleus. IAPs are believed to restrain basal caspase activity in cells (43). Therefore, the intranuclear localization of some IAPs could be part of a regulatory mechanism whereby any active caspases in the nucleus are checked. However, it is also likely that the intranuclear IAPs have functions different from inhibition of caspases. For example, Survivin, an IAP family member, localizes to the nucleus of normal cells and is known to function both as a regulator of apoptosis and the cell cycle (28, 29).

As reported here, cIAP1 localization changes during mitosis, and that cIAP1 overexpression leads to cell cycle aberrations. Moreover, cIAP1 is capable of interacting with Survivin in vitro, and they also colocalized in vivo in mitotic cells. These data suggest novel functions for cIAP1 in the cell cycle. We speculate that the cytokinesis defects seen in cIAP1-overexpressing cells are due to interference with Survivin functions required for proper chromosome segregation and cytokinesis. The ability of cIAP1 overexpression to interfere with Survivin may also explain why it has been so difficult for investigators to establish cell lines stably expressing cIAP1.

Because cIAP1 has an E3-ubiquitin ligase function via its RING domain, it is possible that some cell cycle components are targeted by cIAP1 for ubiquitination and subsequent degradation. In dividing cells at telophase, we found that cIAP1 concentrates both in the re-formed nuclei and at the midbody. It is now well established that the ubiquitin-mediated degradation of several mitotic proteins, including Survivin, BubR1, and Cyclin B1, is essential for proper mitotic exit and cytokinesis (44–46). The intrinsic ubiquitin ligase functions of cIAP1 and its localization to the midbody during telophase raise the intriguing possibility that cIAP1 could be an integral component of a ubiquitin-ligase system operating at a precise time and intracellular location for completion of cytokinesis. However, because cIAP1 knockout mice are born normally and have no overt phenotype, it is unlikely that cIAP1 is uniquely required for midbody degradation. The recent report that cIAP1 is exported from the nuclei of differentiating cells also has interesting implications because upon terminal differentiation and cessation of cell division competency, a nuclear role for cIAP1 in regulating cell cycle may no longer be necessary.

In a recent study that used cDNA microarray technology to analyze HeLa cells for cell cycle–based periodicity of expressed genes, the expression patterns for cIAP1 (BIRC2) and Survivin (BIRC5) were found to be cell cycle–dependent at the mRNA level (47). Whereas the cell cycle–dependent expression of Survivin has already been established, the pattern for cIAP1 was described for the first time in the report. However, a change in cIAP1 protein levels with cell cycle was not confirmed in our study by using immunoblotting analysis.

Overexpression of cIAP1 is one of the hallmarks of prostate cancer progression (37), and overexpression of cIAP1 has also been reported in ovarian cancer (48). Our finding of an effect of cIAP1 on cell growth and cytokinesis suggests that overexpression of this protein may contribute to genetic instability associated with cell cycle and checkpoint perturbations, in addition to impacting apoptosis resistance. Thus, cIAP1 may exert several functions that are relevant to tumor biology.

In conclusion, we present evidence showing that cIAP1 is a nuclear protein and it translocates to the cytosol in response to apoptotic signals that activate caspases. The nuclear localization of cIAP1 is dependent on the Bir domains. Our results also indicate that cIAP1 modulates the cell cycle, and overexpression of cIAP1 causes genomic defects because of defective cell division, possibly through interference with Survivin. The mechanisms regulating cIAP1 trafficking into and out of the nucleus and the relevance of cIAP1 to cell cycle regulation remain to be elucidated.


   Acknowledgments
 
Grant support: NIH grant AG15402 and DoD postdoctoral fellowship DAMD17-01-1-0171 (T. Samuel).

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 Judie Valois for assistance in the preparation of this article and Drs.WeiJiang and C.A. Stein for supplying reagents and for helpful discussions.


   Footnotes
 
Note: K. Okada is currently at the Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

Received 8/18/04. Revised 10/ 5/04. Accepted 10/25/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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