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Molecular and Cellular Biology, May 2007, p. 3511-3520, Vol. 27, No. 9
0270-7306/07/$08.00+0     doi:10.1128/MCB.01448-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Interaction of a Cyclin E Fragment with Ku70 Regulates Bax-Mediated Apoptosis{triangledown} ,{dagger}

Suparna Mazumder,1 Dragos Plesca,1,3 Michael Kinter,4 and Alexandru Almasan1,2*

Departments of Cancer Biology,1 Cell Biology, The Lerner Research Institute,4 Department of Radiation Oncology, Cleveland Clinic, Cleveland, Ohio 44195,2 School of Biomedical Sciences, Kent State University, Kent, Ohio 442403

Received 4 August 2006/ Returned for modification 1 September 2006/ Accepted 13 February 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cyclin E/Cdk2 complex plays an essential role in the G1/S cell cycle transition and DNA replication. Earlier we showed that in hematopoietic tumor cells, caspase-mediated cleavage of cyclin E generates p18-cyclin E, which is unable to interact with Cdk2 and therefore plays a role independent of the cell cycle. The expression of a cleavage-resistant cyclin E mutant greatly diminishes apoptosis, indicating the critical role of cyclin E cleavage. p18-cyclin E expression can induce apoptosis or sensitization to apoptotic stimuli in many cell types. Here we identify Ku70 as a specific p18-cyclin E-interacting partner. In hematopoietic tumor cell lines, the association of p18-cyclin E with Ku70 induces the dissociation of Bax from Ku70, followed by Bax activation. This mechanism of Bax activation leads to the amplification of the apoptosis signal in all tumor cell lines examined. N-terminal Ku70 deletion mutants are unable to bind to p18-cyclin E to regulate its apoptotic effect. p18-cyclin E-mediated amplification of apoptosis is dependent on Bax and Ku70 being greatly diminished in Ku70–/– and Bax–/– mouse embryo fibroblasts and in hematopoietic cells where Bax knockdown was achieved by short interfering RNA. The p18-cyclin E/Ku70 and Bax/Ku70 interactions provide a balance between apoptosis and the survival of cells exposed to genotoxic stress.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis is a universal genetic program of cell death in higher eukaryotes that is involved in cellular development and differentiation as well as responses to various types of stress (6). Genotoxic stress, such as ionizing radiation, induces the activation of Bax, which is a member of the Bcl-2 family of proteins. These proteins are critical regulators of apoptosis (5). Bax normally resides in the cytosol or is loosely associated with mitochondria (42), but upon activation it integrates into the mitochondrial membranes to promote apoptosis (34, 38, 48) via cytochrome c release and caspase activation (2, 3, 17). Cells lacking Bak in addition to Bax are resistant to many apoptotic stimuli that act through the disruption of mitochondrial function, including staurosporine, UV radiation, growth factor deprivation, etoposide, cisplatin, and endoplasmic reticulum stress stimuli (20, 24, 47). However, Bax and Bak perform nonredundant functions during cellular responses to infection by certain pathogens (24). Moreover, recently it has become quite clear that certain apoptotic stimuli, such as Myc in fibroblasts in cell culture or in pancreatic ß cells in vivo, require Bax alone for activating the apoptotic pathway (7). Clearly, cell death in specific developmental organs, such as neurons and oocytes, depends on Bax (8, 37). Finally, it has been shown recently that Bax can be sequestered in the cytosol by binding to the C terminus of Ku70, which provides a molecular mechanism for its specific activation (4, 44).

The deregulation of cellular proliferation, together with defects in apoptosis control, are critical for tumor formation (13). Cyclins, in association with their catalytic subunits, the cyclin-dependent kinases, control cell cycle progression by regulating events that drive the transition between cell cycle phases (12, 33, 39). The cyclin E/Cdk2 complex plays an essential and rate-limiting role in the transition between the G1 and S phases of the cell cycle (36, 40) and in the initiation of DNA replication (22, 36). In addition to its role in cell cycle control (11, 25, 36, 40), cyclin E has been shown to have an important function in genotoxic stress-induced apoptosis of tumor cells of hematopoietic origin (30, 31). Cyclin E levels are upregulated by genotoxic stress, and increased cyclin E expression sensitizes cells to apoptotic stimuli (32). Moreover, during apoptosis, cyclin E is proteolytically cleaved to generate a C-terminal p18-cyclin E fragment. Unlike full-length cyclin E, p18-cyclin E cannot interact with Cdk2, suggesting that its apoptotic function is independent of the cyclin E/Cdk2 complex and its associated kinase activity (30).

The major pathway dedicated to the repair of DNA double-strand breaks in mammalian cells is that of nonhomologous end joining (NHEJ) mediated by the Ku70/Ku80 heterodimer, referred to as Ku (29). The nuclear pool of Ku70 is essential for the repair DNA double-strand breaks in vivo as it provides the DNA binding component required for activating the DNA-dependent protein kinase, which is one of the main functions of Ku (23). However, Ku is a relatively abundant protein that is present in both the cytosol and the nucleus (14). Deregulated Ku70 expression has been reported in a number of cancer cell lines (28). Decreased Ku70 levels can increase cellular sensitivity to stresses that trigger the mitochondrial apoptotic pathway. For example, it was reported that Ku70-deficient cells display increased sensitivity to ionizing radiation (18, 28). Ku70 can also function as a physiological cytoprotector by binding Bax and sequestering it in the cytosol, thereby preventing Bax activation and subsequent apoptosis (4, 44).

In this study, we identify Ku70 as the critical interacting partner of p18-cyclin E. As a result of this interaction, Bax dissociates from Ku70 in hematopoietic cells that are undergoing apoptosis following genotoxic stress or in cells where p18-cyclin E is expressed ectopically. These findings identify a novel mechanism of amplification of cell death through a key step in Bax activation that is critical for apoptosis induced by genotoxic stress.

(This work has been presented partly at the CSHL Meeting on Programmed Cell Death [September 2005] and the International Symposium on the Mechanism of Cell Death [June 2006].)


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and treatments. The IM-9 and NCI-H929 B cell-derived and the Molt-4 T-cell-derived tumor cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI medium with 10% fetal bovine serum (BioWhittaker), L-glutamine, and 100 units/ml penicillin-streptomycin. HEK293T human kidney cells, NCI-H1299 human lung carcinoma cells (also from the ATCC) and Ku70+/+, Ku70–/– (41), Bax–/– (52), and cyclin E1/cyclin E2–/– (16) mouse embryo fibroblasts (MEFs) (generously provided by D. Boothman, S. Matsuyama, C. B. Thompson, and P. Sicinski) were maintained in Dulbecco's modified Eagle's medium (Gibco-BRL) containing 10% fetal bovine serum and antibiotics. Cells (2 x 105/ml) were irradiated with 10 Gy, unless otherwise indicated (137Cs source; fixed dose rate of 2.8 Gy/min) (17). A broad-spectrum, pan-caspase inhibitor (zVAD-fmk), specific cell-permeable caspase-2 (VDVAD-fmk) and caspase-3 (DEVD-fmk) inhibitors, and all pNA-based caspase substrates were obtained from Calbiochem. The caspase-9 inhibitor LEHD-fmk was obtained from Invitrogen. All other chemicals were from Sigma Chemicals (St. Louis, MO) or Invitrogen, unless otherwise noted.

Transfection studies. Cells were transiently transfected with various Ku70 or cyclin E constructs using Lipofectamine (Invitrogen) as described previously (30). Bax knockdown was achieved in IM-9 cells using siLentFect lipid reagent (Bio-Rad) and siBax oligonucleotides (siGENOME SMART pool; Dharmacon) as described previously (38).

Western blot and immunoprecipitation analysis. For immunoblots, cells were lysed in a buffer consisting of 20 mM HEPES, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% NP-40, 1 mM dithiothreitol, and protease inhibitors and immunoblotting was performed with antihemagglutinin (anti-HA) (1:1,000), anti-His (1:500; Covance, Berkeley, CA), anti-Flag (1:500; Sigma), anti-Bax (6A7) (1:1,000; Pharmingen, San Diego, CA), anti-poly(ADP-ribose) polymerase 1 (anti-PARP-1) (1:1,000), anti-active caspase-3 (1:1,000; Cell Signaling, Beverly, MA), anti-ß-actin (1:2,000; Sigma), anti-Ku70 (1:250), and anti-Ku80 (1:250; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. For immunoprecipitation, the lysis buffer composition was similar to that employed for Western blots except that 0.05% NP-40 was used instead of 1%. Bax immunoprecipitation was performed with a polyclonal antibody to Bax (Santa Cruz) in 1% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} buffer containing freshly added protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and leupeptin). Precleared cell lysates (500 µg) were incubated with 0.5 µg antibody for 2 h at 4°C, followed by incubation with protein A plus G agarose beads for 1 h at 4°C. Beads were washed with lysis buffer and boiled with 2x sample buffer. The eluted proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting was performed using the appropriate antibodies, as described previously (30).

Apoptosis assays. Apoptosis was determined by examining the generation of active caspase-3, active Bax, and the 85-kDa PARP-1-derived proteolytic fragment in Western blot studies with antibodies for active caspase-3, active Bax (6A7), and PARP-1. In some experiments, the loss of cell viability was directly assessed by trypan blue exclusion by light microscopy. Two hundred cells were counted for each experiment. Each experimental data point, indicating the percentage of cells undergoing apoptosis, represents the mean ± standard error of the mean (SEM) of results from two experiments. Caspase assays were performed for colorimetric detection (405-nm absorbance) by using VDVAD-pNA, DEVD-pNA, and LEHD-pNA as substrates for caspase-2, -3, and -9, respectively, as described previously (3, 17).

Yeast two-hybrid screening. p18-cyclin E (as bait) was subcloned following the GAL4 DNA binding domain of the pGBKT7 vector containing a c-Myc epitope tag (MATCHMAKER two-hybrid system 3; Clontech, CA). The K562 hematopoietic cell cDNA library was cloned following the GAL4 DNA activation domain of the pACT2 vector (HA epitope tag). When bait and library fusion proteins interact, transcriptional activation of the reporter genes is expected. The Saccharomyces cerevisiae AH109 yeast strain containing the ADE2 and HIS3 markers was cotransformed with the DNA binding domain (bait) and the activation domain (fusion library) (at a titer of ~2 x 106 CFU) containing the TRP1 and LEU2 selection markers, respectively. Yeast growth, selection, and screening were performed following the manufacturer's instructions (MATCHMAKER two-hybrid system 3; Clontech, CA).

Mass spectrometry. HEK293T cells were transiently transfected with cyclin E and p18-cyclin E. At 16 h posttransfection, cell lysates were subjected to immunoprecipitation with an anti-HA antibody, as these constructs have an expressed HA tag at the C terminus. The immunoprecipitates were separated by SDS-PAGE, transferred to membrane, and stained with Coomassie blue, and specific bands were analyzed by mass spectrometry. For protein digestion, the bands were cut from the gel as closely as possible to minimize excess polyacrylamide and washed/destained in two aliquots of 50% ethanol-5% acetic acid. The gel pieces were washed in 0.1 M ammonium bicarbonate and dehydrated in acetonitrile before reduction with dithiothreitol and alkylation with iodoacetamide. The gel pieces were then dehydrated in acetonitrile, washed in 0.1 M ammonium bicarbonate, dehydrated, and dried with a Speed-Vac. Gel pieces were rehydrated in 30 µl of trypsin (20 ng/µl) in 50 mM ammonium bicarbonate on ice for 10 min. Any excess trypsin solution was removed, and 20 µl of 50 mM ammonium bicarbonate was added. The sample was digested overnight at room temperature. The peptides that were formed were extracted from the polyacrylamide in two aliquots of 30 µl of 50% acetonitrile-5% formic acid. These extracts were combined and evaporated to <20 µl for liquid chromatography-mass spectrometry analysis (Finnigan LCQ-Deca ion trap mass spectrometer system with a Protana microelectrospray ion source interfaced with a Phenomenex Jupiter C18 reverse-phase capillary chromatography column).

Two-microliter volumes of the extract were injected, and the peptides were eluted from the column by an acetonitrile-0.05 M acetic acid gradient. The digest was analyzed using the data-dependent multitask capability of the instrument acquiring full-scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in successive instrument scans. The data were analyzed by using all collision-induced dissociation spectra collected in the experiment to search the NCBI nonredundant database with the search program TurboSEQUEST. All matching spectra were verified by manual interpretation. The interpretation process was also aided by additional searches using the Mascot and FASTA programs.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ku70 is a p18-cyclin E binding protein. We have shown previously that p18-cyclin E is generated by proteolytic cleavage of cyclin E during genotoxic stress-induced apoptosis in hematopoietic cells (30). To investigate the mechanism by which p18-cyclin E participates in cell death, a yeast two-hybrid screen was used to search for p18-cyclin E-interacting partners that might mediate its effect. Of the 54 clones that were isolated as p18-cyclin E-interacting partners (Table 1), one promising candidate that emerged was Ku70, a known critical component of the NHEJ DNA repair pathway. Ku70, the Ku70 binding protein KuB3 (49), and several other clones were found to interact with c-Myc-tagged p18-cyclin E by immunoprecipitating the HA epitope-tagged proteins from yeast lysates, followed by immunoblot analyses with an anti-c-Myc antibody (Fig. 1A). These results were confirmed by reverse coimmunoprecipitation experiments with anti-c-Myc antibody followed by Western blot analysis with anti-HA antibody (Fig. 1B). Mass spectrometric analyses of anti-HA immunocomplexes from HEK293T cells transfected with HA-p18-cyclin E independently identified Ku70 as a p18-cyclin E-interacting protein (Fig. 1C).


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  		TABLE 1. p18-cyclin E interacts with Ku70a

 

Figure 1
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  		FIG. 1. Identification of p18-cyclin E interaction with Ku70 in yeast two-hybrid assays. (A) p18-cyclin E-interacting proteins (HA tagged) were identified in yeast cell lysates (500 µg) by yeast two-hybrid assays (Table 1) using anti-HA immunoprecipitation (IP) followed by immunoblotting (IB) for c-Myc-tagged p18-cyclin E. (B) Reciprocal immunoprecipitation and immunoblot analyses of Ku70 and KuB3 proteins, respectively, were performed with the respective primary antibodies. (C) Mass spectrometric identification of interacting proteins. Immunoprecipitates of HA-cyclin E and HA-p18-cyclin E-transfected HEK293T cells were separated by SDS-PAGE, and following Coomassie blue staining, the bands marked by "<" and identifying numerals were excised and subjected to mass spectrometry as described in Materials and Methods. Arrows designate immunoglobulin G heavy chain [IgG(h)] and light chain [IgG(l)] and p18-cyclin E used as reference markers.

 
The possible interaction of p18-cyclin E and full-length cyclin E with Ku70 was also confirmed with immunoprecipitates from HA-p18-cyclin E- or HA-cyclin E-expressing HEK293T cells by using antibodies to either Ku70 or HA, followed by Western blot analysis for HA or Ku70 with the corresponding antibodies (Fig. 2A, top left panel, and B). Our results show that even though p18-cyclin E and full-length cyclin E were expressed at comparable levels and there was equal input of Ku70 (Fig. 2A, right and bottom panels), only p18-cyclin E interacted with endogenous Ku70. To further examine the association of Ku70 with p18-cyclin E, HEK293T cells were cotransfected with HA-p18-cyclin E and Flag-tagged wild-type Ku70 (amino acids 1 to 609) or the derivative deletion mutants, Ku70(1-535) (includes amino acids 1 to 535) and Ku70(496-609) (includes amino acids 496 to 609). Anti-Flag immunoprecipitation of HEK293T cell lysates, followed by immunoblotting with antibodies to HA, indicated that p18-cyclin E can interact with full-length Ku70 and Ku70(1-535), but not with the Ku70(496-609) mutant (Fig. 2D), even though all constructs were expressed at comparable levels (Fig. 2C). As expected, in a similar manner, Ku80 also interacted with full-length Ku70 and Ku70(1- 535), but not with the Ku70(496-609) mutant (Fig. 2D). These results were confirmed by immunoprecipitating with anti-HA antibody and Western blotting with anti-Flag antibody (data not shown). Thus, p18-cyclin E appears to interact with an N-terminal, but not the C-terminal, domain of Ku70.


Figure 2
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  		FIG. 2. Identification of p18-cyclin E interaction with the N terminus of Ku70. (A) In vivo interaction of Ku70 with p18-cyclin E was examined in HEK293T cells transiently transfected with HA-cyclin E or HA-p18-cyclin E. At 16 h posttransfection, cells were lysed with a 0.05% NP-40-containing buffer and immunoprecipitation (IP) (400 µg) was performed with antibodies to Ku70, followed by immunoblot (IB) analyses for HA (left panel). The expression of HA-tagged cyclin E and p18-cyclin E was also examined by immunoprecipitation/immunoblot analyses with anti-HA antibody (right panel). The Ku70 input, determined by the amount of Ku70 that was brought down by immunoprecipitation, was determined by immunoprecipitation/immunoblot analysis with an antibody to Ku70 (bottom panel). (B) Immunoprecipitation (400 µg) was performed with anti-HA antibody, followed by immunoblot analyses for Ku70. (C) To determine the interaction of Ku70 and derivative mutants with p18-cyclin E, HEK293T cells were cotransfected with a Flag-Ku70 construct and derivative deletion mutants together with HA-p18-cyclin E. Cells were collected at 16 h posttransfection and immunoblotted for Flag-tagged Ku70. (D) Immunoprecipitation of Flag-tagged Ku70 was followed by Western blot analysis for Ku80 and p18-cyclin E using antibodies to HA. IgG(h), immunoglobulin G heavy chain; IgG(l), immunoglobulin G light chain.

 
In vitro binding of p18-cyclin E with Ku70 mutants. To determine the specific domain of Ku70 that is critical for its interaction with p18-cyclin E, deletion mutants were generated by mutagenesis (Fig. 3A). Recombinant His-Ku70 and derivative deletion mutants were examined for their abilities to interact in an in vitro interaction assay with recombinant glutathione S-transferase (GST)-p18-cyclin E (Fig. 3B). All proteins were expressed at similar levels (Fig. 3C). Under these conditions, full-length Ku70 and Ku70(1-535) and Ku70(136-535) deletion mutants interacted efficiently with p18-cyclin E, although the interaction of the Ku(136-535) mutant may have been weaker. In contrast, there was no detectable interaction with any of the other mutants, which included mutants with N-terminal Ku70(236-535) and Ku70(336-535) deletion mutants (Fig. 3B). These data indicate that the Ku70 domain of amino acids 136 to 235 is critical for the interaction with p18-cyclin E. Therefore, we conclude that the domain of Ku70 that is essential for p18-cyclin E interaction is located in its N-terminal domain and is distinct from the Bax-interacting domain (amino acids 536 to 609).


Figure 3
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  		FIG. 3. In vitro binding of p18-cyclin E to Ku70 and derivative mutants. (A) Diagram of the recombinant Ku70 deletion mutants. (B) Full-length Ku70 and five deletion mutants were expressed in pET32A. Bacterial lysates were immunoblotted with anti-His antibodies after pulldown with anti-GST from an in vitro mixing experiment with GST-p18-cyclin E. (C) Immunoblotting of bacterial lysates with anti-His antibody was used as a control. The asterisk designates the His-Ku70 and derivative deletion mutants.

 
Interaction of p18-cyclin E with Ku70 induces the disruption of the Bax/Ku70 complex and cell death. To characterize the biological significance of p18-cyclin E binding to Ku70, we examined its effect on the interaction of Ku70 with Bax. HEK293T cells were transfected with p18-cyclin E used alone or in combination with different Ku70 mutants, and cell lysates were immunoprecipitated for Bax and immunoblotted for Ku70 (Fig. 4A). The association of endogenous Bax with Ku70 was diminished ~60% by the overexpression of p18-cyclin E (Fig. 4A, lane 2). Cell death, as measured by trypan blue exclusion, was apparent in about 30% of cells following p18-cyclin E expression, confirming that p18-cyclin E can induce cell death when expressed ectopically. Cell death and Bax activation (Fig. 4B and C) caused by p18-cyclin E correlated with a reduction in the Bax/Ku70 association (Fig. 4A). p18-cyclin E-induced loss of cell viability, as well as Bax activation, could be prevented by coexpression of wild-type Ku70 or the Ku70(1-535) but not Ku70(496-609) mutant (Fig. 4B and C). Strikingly, Ku70(496-609) could not reverse Bax activation by p18-cyclin E, leading to diminished association of Ku70 with Bax. This is because once Bax gets activated following its displacement from Ku70 by p18-cyclin E, active Bax can no longer bind to Ku70(496-609) (Fig. 4A and C). However, Ku70(496-609) and wild-type Ku70 can abolish the cell death induced by green fluorescent protein-Bax (see the supplemental material), as the Ku70 domain of 496 to 609 amino acid residues binds to inactive Bax in the cytosol (4). Moreover, we have tested these mutants in cells that do not express p18-cyclin E. The expression of Ku70-derivative mutants alone does not have a significant effect on cell death. From the results of these experiments, we conclude that Bax is present in a complex with Ku70, and a disruption of this complex by p18-cyclin E correlates with increased apoptosis, suggesting that Bax mediates the apoptotic effect of p18-cyclin E although there could be a possibility of Bax-independent cell death induced by p18-cyclin E.


Figure 4
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  		FIG. 4. p18-cyclin E-induced apoptosis involves dissociation of Bax from Ku70. (A) HEK293T cells were transfected with Flag-Ku70 and derivative deletion mutants and HA-p18-cyclin E, alone or in combination, as indicated. At 16 h posttransfection, cells were lysed with 1% CHAPS buffer, immunoprecipitated (IP) with an anti-Bax antibody, and immunoblotted (IB) for Ku70. Percent inhibition of the Bax/Ku70 association is indicated as mean values ± SEM from three independent experiments. (B) HEK293T cells were transfected as described above. Loss of cell viability was examined at 16 h posttransfection by trypan blue exclusion and scored as marked in the graphs. Data shown are mean values ± SEM from three independent experiments. Error bars indicate standard deviations. (C) HEK293T cells were transfected as described above. At 16 h posttransfection, cells were lysed with 1% CHAPS buffer, immunoprecipitated with an anti-Bax 6A7 antibody, and immunoblotted for active Bax. IgG(h), immunoglobulin G heavy chain; IgG(l), immunoglobulin G light chain.

 
Ku70 and Bax are required for the apoptotic function of p18-cyclin E independent of cyclin E. To further investigate whether Ku70 plays a direct role in mediating p18-cyclin E-induced apoptosis, p18-cyclin E was transiently overexpressed in fibroblasts obtained from wild-type and Ku70-deficient mouse embryos (41). p18-cyclin E expression induced Bax activation and cell death in p18-cyclin E-expressing wild-type MEFs. In contrast, neither Bax activation nor significant cell death was observed in Ku70–/– MEFs (Fig. 5A, top and middle panels).


Figure 5
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  		FIG. 5. Ku70 and Bax are critical for p18-cyclin E-mediated cell death. (A) p18-cyclin E overexpression activates cell death in Ku70+/+ but not Ku70–/– MEFs. The activation of Bax was examined with an active conformation-specific antibody (6A7). Immunoprecipitation as well as immunoblotting was performed with antibodies to 6A7. Immunoglobulin G heavy chain [IgG(h)] levels from the same blot served as a loading control (top panel). Cell death was indicated by trypan blue exclusion. Data shown are the mean values ± SEM from two independent duplicate experiments (middle panel). Error bars indicate standard deviations. The expression of p18-cyclin E was examined by anti-HA (bottom panel). (B) Bax+/+ as well as Bax–/– MEFs were transfected with different concentrations of p18-cyclin E DNA (2 and 4 µg). At 16 h posttransfection, cells were immunoprecipitated for Bax, followed by immunoblotting for active Bax with the 6A7-specific antibody. The activation of Bax in wild-type MEFs is shown in the top panel, cell death by trypan blue exclusion in the middle panel, and HA-p18-cyclin E expression in the bottom panel. (C) IM-9 cells untreated (–) or treated (+) with siRNA oligonucleotides targeting Bax were transfected with HA-p18-cyclin E. Bax knockdown was achieved by transient transfection with 100 nM siBax, which was added sequentially every 24 h. At 24 h posttransfection with p18-cyclin E and at 24 or 48 h after the introduction of siBax, cells were immunoblotted for Bax, PARP-1, and HA-tagged p18-cyclin E. ß-actin was used as a loading control. (D) cyclin E1/E2+/+ and cyclin E1/E2–/– MEFs were transfected with p18-cyclin E and immunoblotted to evaluate activation of caspase-3, PARP-1 cleavage, and p18-cyclin E expression. GFP, green fluorescent protein.

 
To directly demonstrate the role of Bax in p18-cyclin E-mediated cell death, similar experiments were performed with wild-type and Bax-deficient MEFs (52). In wild-type mouse cells, both Bax activation and the extent of cell death were p18-cyclin E dose dependent (Fig. 5B, top and middle panels). While p18-cyclin E induced an increase of up to sixfold in levels of cell death in wild-type MEFs, there was little change (less than twofold) in the extent of apoptosis in Bax–/– MEFs (Fig. 5B, middle panel). To further demonstrate the contribution of Bax to p18-cyclin E-mediated cell death in human hematopoietic cells, where robust cyclin E cleavage can take place, p18-cyclin E was expressed in IM-9 B-cell-derived tumor cells in which RNA interference was used to diminish Bax levels. Apoptosis, as determined by PARP-1 cleavage, was completely prevented in the IM-9 cells where Bax levels were depleted by specific short interfering RNA (siRNA) oligonucleotides (Fig. 5C). Taken together, these data indicate that both Bax and Ku70 are required for p18-cyclin E-mediated apoptosis to occur.

Cyclin E has a well-known role in cell cycle regulation. However, no interaction was detected between p18-cyclin E and Cdk2 by transfection-based assays, suggesting that p18-cyclin E has no effect on its cyclin E/Cdk2-associated function (30). To directly address whether the functional contribution of p18-cyclin E to apoptosis is indeed independent of the cell cycle regulatory function of cyclin E, p18-cyclin E was expressed in wild-type and cyclin E knockout MEFs. As human and mouse cells express two cyclin E genes, cyclin E1 and cyclin E2, both have been inactivated by homologous recombination to prevent any possible compensatory mechanisms (16). p18-cyclin E induced robust apoptosis as determined by PARP-1 cleavage and the activation of caspase-3 in both wild-type and cyclin E1/E2 knockout MEFs, indicating that the cell death-inducing effect of p18-cyclin E is independent of the well-known cell cycle regulatory function of cyclin E (Fig. 5D). Interestingly, there seems to be less caspase activation in cyclin E+/+than in cyclin E–/– cells (Fig. 5D). Nevertheless, cell death mediated by p18-cyclin E takes place even in the absence of cyclin E. This is important as our (32) and other (21) reports indicate that cyclin E/Cdk2 can also participate in cell death, a component that can be ruled out in the present study. There is not only more active caspase-3 but also cleaved PARP-1, the latter likely being attributed to higher levels of constitutive PARP-1 expression in these cells. The reason for more PARP-1 and caspase-3 proteolytic cleavage in cyclin E-deficient cells is unclear.

p18-cyclin E generation in hematopoietic cells is essential for dissociation of Bax from Ku70 and its activation. We next examined the complex formation of Ku70 with Bax in B-cell-derived IM-9 tumor cells. We previously reported that these cells undergo apoptosis when challenged with apoptotic stimuli, such as ionizing radiation or DNA-damaging chemotherapeutic agents (3, 30). A coimmunoprecipitation experiment indicated that Bax associates with Ku70 in IM-9 cells (Fig. 6A). This interaction was diminished at 16 and 24 h after irradiation. The p18-cyclin E that was generated following irradiation (Fig. 6B) associated with Ku70 (Fig. 6C), with most of the endogenous p18-cyclin E being found in this complex. The p18-cyclin E binding to Ku70 coincided with the dissociation of Bax from Ku70 and Bax activation, as determined by its immunoreactivity with the active Bax conformation-specific antibodies (Fig. 6A). These events were preceded by the activation of caspase-3 and the cleavage of its cellular substrates: PARP-1 and cyclin E (Fig. 6B). Although caspase-3 activation and PARP-1 cleavage were apparent at 4 h, there was a minimal fraction of p18-cyclin E present at this time. By 8 h, however, levels of p18-cyclin E became more abundant. Concomitantly, levels of cyclin E decreased dramatically by 24 h as p18-cyclin E levels rose. These data support the idea of a sequential activation of caspases and cleavage of cyclin E to generate p18-cyclin E, which is followed by Bax dissociation from Ku70 and its activation.


Figure 6
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  		FIG. 6. Sequential p18-cyclin E generation, release of Bax from the Bax-Ku70 complex, and Bax activation in hematopoietic cells. IM-9 cells were collected at the indicated time points following irradiation. (A) Cells were lysed with 1% CHAPS buffer and immunoprecipitated (IP) with anti-Bax antibody, followed by Western blot analysis for Ku70, or alternatively immunoprecipitated and immunoblotted (IB) with the 6A7 antibody for active Bax protein. (B) Cells were lysed with 1% NP-40 lysis buffer and analyzed by immunoblotting with the corresponding primary antibodies for p18-cyclin E generation, activation of caspase-3, and PARP-1 cleavage following irradiation. ß-Actin was used as a loading control. The asterisk designates a cross-reactive band. (C) Cells were lysed with 0.05% NP-40 lysis buffer. Immunoprecipitation was then performed for cyclin E, and Western blot analysis was performed for Ku70 protein. (D) IM-9 cells treated with zVAD-fmk (100 µM) at 0 h or 8 h postirradiation were immunoprecipitated with an anti-Bax antibody and immunoblotted for Ku70. (E) IM-9 cells stably expressing Bcl-2 were examined at 48 h following irradiation for the presence of Ku70 in Bax immunoprecipitates using the corresponding primary antibodies. IgG(h), immunoglobulin G heavy chain; IgG(l), immunoglobulin G light chain.

 
To ensure that the release of Bax from the Bax/Ku70 complex was due to a direct effect of p18-cyclin E and not to an indirect outcome of apoptosis, IM-9 cells were treated with the broad-spectrum, pan-caspase inhibitor zVAD-fmk at two different time points: either at the time of irradiation (0 h) or at 8 h following irradiation, with cells being examined at 24 h postirradiation. zVAD-fmk prevented cell death to a large extent when it was added either before or at 8 h following irradiation (3). However, zVAD-fmk could not prevent Bax dissociation from Ku70 when it was added at 8 h (Fig. 6D) as p18-cyclin E appeared within 8 h following irradiation.

A well-established molecular mechanism to inhibit Bax-induced apoptosis is through Bcl-2 overexpression (10). We have previously shown that ectopic expression of Bcl-2 in IM-9 cells not only prevents apoptosis (3) but also inhibits the appearance of p18-cyclin E (30). The examination of the Bax/Ku70 association indicates that Bcl-2 expression also precludes any changes in the Bax/Ku70 association, even at 48 h following irradiation (Fig. 6E). This result, taken together with the above data, indicates that when p18-cyclin E genesis is prevented by blocking caspase activation or by Bcl-2 expression, there is no change in the Bax/Ku70 interaction following irradiation. This indicates that the disruption of the Ku70/Bax interaction following irradiation of hematopoietic tumor cells is not a consequence of apoptosis and that the dissociation of Bax from Ku70 requires p18-cyclin E.

Caspase-9 but not caspase-2 is required for p18-cyclin E genesis. Earlier we showed that the generation of p18-cyclin E is mediated by caspase-3 or a caspase-3-like activity (30). To further delineate which upstream caspase is involved in cyclin E cleavage, IM-9 cells were treated with specific inhibitors of caspase-9, caspase-3, and caspase-2 as well as the broad-spectrum inhibitor (zVAD-fmk) either at the time of irradiation (0 h) or at 8 h following radiation treatment, with cells being examined at 24 h postirradiation. While zVAD-fmk and the caspase-3 and caspase-9 inhibitors were effective (when added at the time of irradiation [0 h]) in preventing cell death (Fig. 7A) and the generation of p18-cyclin E and, as a consequence, decreased cyclin E levels (Fig. 7B), the caspase-2 inhibitor VDVAD-fmk had no effect. The proportion of cells that lost viability following irradiation was ~70%, whether or not the caspase-2 inhibitor was present, with less than 5% of cells being nonviable when any of the other three caspase inhibitors were used. However, while the addition of zVAD-fmk and the caspase-3 and caspase-9 inhibitors at 8 h following irradiation still partially blocked the loss of cell viability, it did not have any effect on the generation of p18-cyclin E and the concomitant decrease in cyclin E levels, indicating that cyclin E cleavage is preceding full activation of apoptosis. Consistent with the results with caspase inhibitors, there was very limited caspase-2 activity, with much more robust caspase-9 and caspase-3 activities by 4 h following irradiation. Caspase-2 levels continued to remain minimal, with levels of caspase-9 and -3 activity remaining steady up to 16 h, a time after which all caspases became more active (Fig. 7C). Using lysates from the experiment shown in panel B indicated that the caspase-2 inhibitor VDVAD-fmk was nevertheless effective in blocking most (>70%) of the residual caspase-2 activity with no effect on caspase-9 (data not shown). These data indicate that caspase-9, not caspase-2, is the initiator caspase in the radiation-induced apoptosis of these cells that leads to caspase-3 activation, cyclin E cleavage, and subsequent Bax activation. A schematic model depicts the key pathways for genotoxic stress-induced apoptosis through the activation of caspases-9 and -3, with p18-cyclin E providing an amplification step required for Bax translocation to mitochondria (Fig. 7D).


Figure 7
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  		FIG. 7. Caspase-9 and -3 activation are required for generation of p18-cyclin E and apoptosis. (A) Loss of cell viability (trypan blue exclusion) of IM-9 cells was determined by the addition of the pan-caspase inhibitor zVAD-fmk or specific inhibitors (VDVAD-fmk, DEVD-fmk, and LEHD-fmk) of caspase-2, caspase-3, and caspase-9 at 0 h or at 8 h following irradiation, with cells being examined at 24 h postirradiation. Error bars indicate standard deviations. (B) Western blot analysis was performed with the lysates of the samples as described in panel A to determine the levels of cyclin E and p18-cyclin E. (C) Caspase assays with colorimetric substrates for caspase-2, caspase-3, and caspase-9 were performed with IM-9 cells collected at the indicated time points following irradiation. (D) A model for the role of p18-cyclin E in an amplification stage required for the intrinsic pathway of apoptosis. Ku70 prevents Bax activation by sequestering it into the cytosol. The activation of caspase-9 and then caspase-3 leads to proteolytic cleavage of cyclin E at the Asp D275 position (30) to generate the p18-cyclin E fragment that dissociates Bax from Ku70, leading to Bax activation and its translocation to mitochondria. The mitochondrion-dependent apoptotic events and cyclin E cleavage can be blocked by Bcl-2.

 
p18-cyclin E-mediated dissociation of Bax from Ku70 triggers apoptosis. Earlier we showed that p18-cyclin E is produced following the irradiation of other hematopoietic tumor cell lines, such as Molt-4 T-cell lymphoma (30). Similar to the results obtained with IM-9, Bax dissociated from Ku70 following the irradiation of Molt-4 as well as NCI-H929 multiple myeloma cells (Fig. 8A), indicating the generality of the interaction of Bax with Ku70 in hematopoietic tumor cell lines and its disruption following irradiation. While Molt-4 cells are known to be sensitive, NCI-H929 cells are quite resistant to radiation, indicating that cyclin E cleavage and Bax dissociation take place regardless of cellular radiation sensitivity (Fig. 8A).


Figure 8
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  		FIG. 8. p18-cyclin E-mediated dissociation of Bax from Ku70 enhances apoptosis. (A) Bax immunoprecipitation (IP) and Ku70 immunoblot (IB) analyses were performed for NCI-H929 multiple myeloma and Molt-4 T-cell lymphoma cells, following irradiation with 10 and 4 Gy for 48 and 18 h, respectively, as described in the legend for Fig. 5. (B) NCI-H1299 cells were irradiated (10 Gy) with or without transient expression of HA-p18-cyclin E. The extent of apoptosis was determined by examining active caspase-3 generation and PARP-1 cleavage in cells collected at 24 h posttransfection. ß-Actin was used as a loading control. (C) NCI-H1299 cells expressing HA-p18-cyclin E were lysed with 1% CHAPS buffer and immunoprecipitated with antibodies to Bax and immunoblotted for Ku70 protein to determine the extent of Bax association with Ku70 (left panel). NCI-H1299 cells expressing HA-p18-cyclin E were lysed with 0.05% NP-40 lysis buffer, immunoprecipitated with anti-HA antibody, and immunoblotted for Ku70 to examine the association of p18-cyclin E with Ku70 (right panel). IgG(h), immunoglobulin G heavy chain; IgG(l), immunoglobulin G light chain.

 
To further examine whether p18-cyclin E expression can overcome radiation resistance, the response of NCI-H1299 lung tumor-derived epithelial cells, which are also known to be resistant to ionizing radiation (35), was examined. Indeed, there was very limited apoptosis following irradiation, as determined by the activation of caspase-3 and the cleavage of its cellular substrate PARP-1. p18-cyclin E expression resulted in a modest caspase-3 and PARP-1 cleavage. The expression of p18-cyclin E, however, lead to increased apoptosis following subsequent irradiation (Fig. 8B), as indicated by enhanced caspase-3 and PARP-1 cleavage. As expected, the interaction of p18-cyclin E with Ku70 (Fig. 8C, right panel) also induced the disruption of the Bax/Ku70 complex in these cells, as indicated by the Ku70 levels that were found in the Bax immunoprecipitates (Fig. 8C, left panel). These results suggest that the Bax dissociation from Ku70 that is induced by p18-cyclin E enhances the extent of radiation-induced apoptosis.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have investigated the mechanism by which a proteolytic fragment of cyclin E contributes to genotoxic stress-induced apoptosis. This 18-kDa cyclin E fragment that is generated by caspase-mediated proteolytic cleavage is associated with apoptosis in all human tumor hematopoietic cell lines that have been examined so far (30). Cyclin E cleavage results in the abrogation of its binding to Cdk2 and, as determined by transfection assays (30) or with cyclin E-deficient MEFs (Fig. 5), therefore, inactivation of its cell cycle function. Interestingly, it appears that p18-cyclin E may not be generated in other cell types under similar conditions; however, it can induce apoptosis in these cells when it is expressed ectopically. This may not be surprising given that there are hundreds of known caspase substrates, only some of which may be activated in a given cell. Various mechanisms may account for such a selective proteolytic cleavage, such as subcellular compartmentalization of caspases and their substrates, the protection of substrates by scaffolding molecules or other inhibitory molecules, or posttranslational modifications of the caspases or their targets (15). Moreover, while caspases play a central role in most cell death pathways leading to apoptosis, growing evidence also implicates them in nonapoptotic functions (27). Caspases are required for terminal differentiation of specific cell types, such as that during erythropoiesis. Physiological caspase activation is recognized widely as a functional requirement, for example, for erythroid maturation through cleavage of the transcription factors GATA-1 and TAL-1 (9, 50). Such nonapoptotic functions of caspases reinforce the notion that cells can restrict the proteolytic activities of these enzymes to selected substrates.

Proteolytic cleavage of cyclin E is critical for its biological function as the expression of a cleavage-resistant cyclin E mutant in hematopoietic cells greatly inhibits genotoxic stress-induced apoptosis (30). In this study, we identify Ku70 as a p18-cyclin E binding partner and examine the role of this interaction in the mechanism of apoptosis. A key regulatory step in the intrinsic cell death pathway is the activation of the proapoptotic molecule Bax (5). Following its activation, Bax integrates into the outer mitochondrial membrane, where it forms pore-like structures by oligomerization, leading to the release of cytochrome c and other death-inducing factors (6, 38, 48). It has been well established that following its exposure to various cytotoxic stimuli, Bax undergoes multiple conformational changes prior to committing cells to dying by permeabilization of their internal membranes, both in mitochondria and the endoplasmic reticulum (43). Although the mechanism of activation of Bax remains unclear, a recent study indicates that Bax is kept inactive in some resistant cells by interacting with cytosolic Ku70 (4). The overexpression of Ku70 prevents Bax-mediated apoptosis, whereas the depletion of Ku70 causes cells to become more sensitive to a variety of apoptotic stimuli. These results demonstrate that in addition to its well-known role in DNA repair, Ku70 is a physiological inhibitor of Bax-induced apoptosis. Interestingly, cells from Ku70 knockout mice are hypersensitive to agents, such as staurosporine, that induce apoptosis in the absence of DNA damage (1). This suggests that Ku70 plays a role in suppressing apoptosis that is independent of its role in DNA repair. Based on these findings, nuclear Ku70 could therefore be considered to be responsible mainly for the repair of DNA damage, whereas the cytosolic pool of Ku70 may be primarily a regulator of Bax activation (4, 44).

We identified Ku70 and characterized its role in regulating Bax-mediated apoptosis in hematopoietic cells that generate p18-cyclin E from cyclin E by limited activation of caspase-3 following genotoxic stress. Bax may be activated at a minimal level by other BH3 proteins to initiate the cell death process. However, by binding to Ku70, p18-cyclin E can cause the dissociation of inactive Bax from Ku70 and its robust activation, thus leading to a feedback amplification loop that further activates apoptosis. Therefore, Ku70 plays a dual role in NHEJ and the regulation of Bax, which in turn may determine the balance between cell survival and death in hematopoietic cells. However, Ku70 may not be the only molecule that sequesters Bax and prevents its activation. Ku70-deficient cells are viable, although somewhat more sensitive to apoptosis, indicating that Bax should be largely inactive even in the absence of its binding to Ku70. This may be explained by the fact that Bax release from Ku70 represents only a first phase in a multistep process required for the activation of Bax. It is also possible that, in addition to Ku70, other mechanisms exist for keeping Bax sequestered and inactive, for example, through its binding to the humanin protein (19, 51).

Our data reveal a novel mechanism of apoptosis by which the proteolytic cleavage of cyclin E regulates the subsequent amplification stage of the intrinsic pathway of apoptosis. Earlier we showed that cyclin E is cleaved by activated caspase-3 to generate p18-cyclin E (30). The cleavage of cyclin E as well as cell death can be prevented by Bcl-2 overexpression (30), indicating that the function of Bcl-2 is upstream of the activation of the mitochondrial apoptotic cascade and cyclin E cleavage. Here, we show that interaction of p18-cyclin E with Ku70, resulting in the liberation of Bax from the Ku70-Bax complex, has an important function in the amplification stage of apoptosis in hematopoietic cells by initiating a robust activation of Bax, which sustains a feedback loop targeting mitochondria (Fig. 7D). These data are consistent with the feedback amplification loop model we have proposed earlier for these cells to link caspase activation to mitochondrial dysfunction in genotoxic stress-induced apoptosis. We have shown that an initial limited, early caspase activation was not sufficient to release enough cytochrome c to lead to significant apoptosis in hematopoietic cells (3). We have now determined that caspase-9, not caspase-2, was involved in the caspase-3 activation that was responsible for the proteolytic cleavage of cyclin E. Previously we have also shown that caspase-8 and -6 inhibition had no effect on p18-cyclin E generation (30).

Recent findings have suggested that caspase-2 can be an apical regulator in apoptotic pathways leading from DNA damage to the release of mitochondrial cytochrome c and subsequent activation of effector caspases. However, a recent report found that caspase-2 activation-mediated apoptosis was observed only in cells with functional p53 (46), consistent with the mechanisms of its activation that involve the p53-dependent formation of the PIDDosome (45). However, p18-cyclin E generation (e.g., in U266 [26]) and apoptotic function (e.g., in H1299 cells) (Fig. 8) are unaltered in p53-deficient cells, including IM-9 with p53 knockdown by siRNA (unpublished data), and therefore, a lack of requirement for caspase-2 is not surprising.

Recently, based on genetic data, it has also been reported that an amplification loop involving mitochondria is required to trigger massive cytochrome c release from mitochondria into the cytosol and abundant effector caspase-3 and/or caspase-7 activation, leading to cell death (26). Consistent with our model, Bax translocation to the mitochondria and cytochrome c release were impaired in cells deficient in effector caspase-3 and -7, indicating that caspases regulate mitochondrial events. This is consistent with a recent account of the mechanism of the cytochrome c role in apoptosis that clearly documents its biphasic release in the cytosol in multiple biologically relevant situations (15).

While p18-cyclin E activates an apoptosis pathway that is dependent on Bax and Ku70, it is possible that p18-cyclin E may also regulate cell death to some extent by a Bax- and/or Ku70-independent pathway. Indeed, neither Bax nor Ku70 deficiency in knockout MEFs nor siRNA-mediated silencing of Bax in IM-9 completely abolished cell death, suggesting a Bax- and/or Ku70-independent apoptosis component induced by p18-cyclin E. Moreover, Ku70 or Ku70(1-535) does not prevent the cell death mediated by p18-cyclin E completely either, indicating the presence of Ku70-independent cell death. Most importantly, once Bax is activated by p18-cyclin E or other stimuli (4, 44), leading to its translocation to mitochondria, the activation cannot be reversed by Ku70(496-609), even though the 496 to 609 amino acid residues of Ku70 bind to the endogenous inactive Bax in the cytosol. This result suggests that a Bax-independent cell death pathway is also present in this system in addition to the predominant Bax-dependent apoptosis.

In conclusion, we have identified p18-cyclin E, Ku70, and Bax as key regulators of the intrinsic apoptotic pathway execution. So far, we have found that cyclin E cleavage is limited to human hematopoietic tumor cells, with their sensitivity to genotoxic stress accounted for by their abilities to generate p18-cyclin E. In cells with functional Ku70 proteins, p18-cyclin E induces the dissociation of Bax from Ku70, which is followed by Bax activation and apoptosis. Moreover, p18-cyclin E-mediated Bax dissociation from Ku70 enhances radiation-induced cell death. p18-cyclin E may thus provide a critical physiological balance between the two key cellular responses to DNA damage (cell cycle control and apoptosis) (13) that, when perturbed, facilitate cancer. Future experiments will determine whether p18-cyclin E binding to Ku70 also impacts the third key cellular response to repair the DNA damage caused by genotoxic stress. Cyclin E expression is increased in several human carcinomas, including those of hematopoietic origin. Our findings suggest that cyclin E-derived fragments, such as p18-cyclin E or smaller derivatives, may become effective therapeutics for the treatment of hematopoietic malignancies.


   ACKNOWLEDGMENTS
 
We thank David Boothman (University of Texas Southwestern Medical Center, Dallas, TX), Susan Lees-Miller (University of Calgary, Calgary, Canada), Craig Thompson (The Abramson Family Cancer Institute, Philadelphia, PA), Piotr Sicinski (Harvard Medical School, Boston, MA), and Shigemi Matsuyama (Case Western Reserve University, Cleveland, OH) for valuable reagents. We also thank Patricia Stanhope-Baker, Amy Moore, Marcela Oancea, and Meredith Crosby for critically reading the manuscript.

This work was supported by research grants from the U.S. National Institutes of Health (CA82858 and CA81504).


   FOOTNOTES
 
* Corresponding author. Mailing address: Cleveland Clinic, Department of Cancer Biology, Cleveland, OH 44195. Phone: (216) 444-9970. Fax: (216) 445-6269. E-mail: almasaa{at}ccf.org Back

{triangledown} Published ahead of print on 26 February 2007. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back


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Molecular and Cellular Biology, May 2007, p. 3511-3520, Vol. 27, No. 9
0270-7306/07/$08.00+0     doi:10.1128/MCB.01448-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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