Bcl-2 Controls Caspase Activation Following a p53-dependent Cyclin D1-induced Death Signal*

M.A. Christine PrattDagger and Min-Ying Niu

From the Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada

Received for publication, September 19, 2002, and in revised form, December 6, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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MCF-7 and ZR-75 breast cancer cells infected with an adenovirus constitutively expressing high levels of cyclin D1 demonstrated widespread mitochondrial translocation of Bax and cytochrome c release that was approximately doubled after the addition of all-trans retinoic acid (RA) or Bcl-2 antisense oligonucleotide. By comparison, the percentage of cells in Lac Z virus-infected cultures containing translocated Bax and cytoplasmic cytochrome c was markedly less even after RA treatment. Despite this, RA-treated Lac Z and untreated cyclin D1 virus-infected cultures contained similarly low proportions of cells with active caspase or cells that were permeable to propidium iodide. Bax activation was p53-dependent and accompanied by arrest in G2 phase. Although constitutive Bcl-2 expression prevented Bax activation, it did not alter cyclin D1-induced cell cycle arrest, illustrating the independence of these events. Both RA and antisense Bcl-2 oligonucleotide decreased Bcl-2 protein levels and markedly increased caspase activity and apoptosis in cyclin D1-infected cells. Thus amplified cyclin D1 expression initiates an apoptotic signal inhibited by different levels of cellular Bcl-2 at two points. Whereas high cellular levels of Bcl-2 prevent mitochondrial Bax translocation, lower levels can prevent apoptosis by inhibition of caspase activation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The D-type cyclins govern the activity of cyclin-dependent kinases (CDKs),1 CDK-4 and CDK-6, which phosphorylate targets including the retinoblastoma protein that are responsible for progression through G1 (1, 2). The importance of cyclin D1 in oncogenesis is reflected in numerous tumor types that display amplification and/or overexpression of the cyclin D1 gene (1). On the other hand, high levels of cyclin D1 can also result in growth suppression and apoptosis (reviewed in Refs. 3 and 4). Similar to other protooncogene products such as Myc, high level expression of cyclin D1 elicits an apoptotic response in the absence of trophic factors, although apoptosis can occur even in the presence of serum (5). Thus increased cyclin D1 expression can also present a survival challenge to the cell.

The Bcl-2 family of proteins plays an integral role in control of programmed cell death. There are now numerous members of this family defined by the presence of four conserved alpha -helical Bcl-2 homology domains called BH-1-4. The central role of the Bcl-2 family in the regulation of cell death is control over mitochondrial membrane permeability and release of cytochrome c into the cytoplasm, where it is free to interact with Apaf-1 and caspase-9 to form an active "apoptosome" complex, inducing a cascade of protease activity and ensuing cell death (6). Proapoptotic molecules including Bax and Bak do not have the BH-4 domain, but their activity depends on the presence of the BH-3 domain (7, 8). Bax is a cytoplasmic monomer in thriving cells and undergoes mitochondrial translocation in response to stress or damage signals following a conformational change in the molecule. Cytochrome c release is thought to occur as a result of the formation of large Bax conductance channels in the outer mitochondrial membrane as multimers, alone or in protein/lipid complexes, or in coordination with the voltage-dependent anion channel (9). Although membrane integration of this "activated" Bax requires the hydrophobic C terminus, the translocation/insertion process is also associated with exposure of the normally occluded N terminus (10-12). Experimentally enforced dimerization of Bax results in mitochondrial translocation but no cytochrome c release and death as a result of mitochondrial dysfunction (13). Other types of proapoptotic molecules are the BH3 domain-only proteins including Bid, Bim, Bad, and the p53-regulated NOXA (14). Bid can activate Bax directly, whereas the latter three proteins can be bound and sequestered by Bcl-2 and Bcl-xL, which is thought to represent their major antiapoptotic activity (14).

The caspase family of cysteine proteases plays an integral role in apoptosis. The apoptosome constituent, caspase-9, is the apical enzyme in the mitochondrial death pathway responsible for activation of effector caspases including caspase-2, -3, -6, -7, and -10 (16). Regulation of caspase activity is afforded by a growing family of inhibitor of apoptosis (IAP) gene products, which contain from one to three domains homologous to the initially described baculovirus IAP repeat domain (17, 18). IAP activity may be inhibited by mitochondrial release of the Smac/DIABLO protein, which then allows for full activation of caspase-9 within the Apaf-1 cytochrome c complex (19, 20). Although apoptosis is usually defined as a caspase-dependent process, an inefficient apoptosis or necrotic cell death can also occur as a result of mitochondrial dysfunction (21, 22).

In the present work, we have acutely expressed cyclin D1 by adenoviral-mediated gene transfer into breast cancer cell lines, resulting in G2/M phase arrest, p53-dependent Bax mitochondrial translocation, and cytochrome c release without accompanying caspase activation. The addition of RA or antisense Bcl-2 to these cells markedly increased caspase activation. The results support the notion that Bcl-2 can not only prevent Bax recruitment to the mitochondrial membrane but can also interfere with caspase activation during the course of an apoptotic signal.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cell Culture and Transfection-- MCF-7 cells and ZR-75 human breast cancer cell lines were grown in Dulbecco's modified Eagle's medium (high glucose) at 37 °C/5% CO2 with 5% fetal bovine serum, 1% non-essential amino acids, 110 µg/ml sodium pyruvate, and 10 µg/ml gentamicin. For treatment with RA, 100-mm plates were seeded with 5 × 105 cells and made 1 µM with RA or the equivalent amount of vehicle after attachment. Cells were harvested at the indicated times. To generate stable HPV-16 E6 clones, CMV-HPV-16 E6 was cotransfected with CMV-puromycin into MCF-7 cells using the calcium phosphate precipitate method as described previously (23). Cells were selected in 2 µg/ml puromycin, and individual clones were isolated and expanded and then evaluated for the expression of the E6 gene by Northern analysis of RNA. p53 protein was assessed in E6-positive clones by immunoblot analysis. Two clones were chosen for experimentation based on very low levels of p53 protein as compared with control MCF-7 cells.

Plasmids and Antibodies-- The HPV-16 E6 oncoprotein cDNA was obtained from Peter Howley (Harvard University). Anti-cyclin D1 monoclonal (HD11) and polyclonal (M20), anti-p53 (Pab 240), anti-Bcl-2, and anti-Bax were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Apaf-1 was purchased from Chemicon (Temecula, CA); monoclonal (mAb) anti-Bax 6A7 was purchased from Trevigen (Gaithersburg, MD); anti-cytochrome c monoclonal antibody was purchased from Pharmingen; and polyclonal antibody (H-104) was obtained from Santa Cruz Biotechnology. Anti-cytochrome c oxidase was purchased from Molecular Probes (Eugene, OR); anti-mitochondrial hsp 70 was purchased from ABR (Golden, CO); anti-Smac/DIABLO was purchased from Prosci (Poway, CA); and secondary antibodies conjugated to horseradish peroxidase, CY3, and fluorescein were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).

Viral Infection-- Lac Z-expressing adenovirus was obtained from Dr. Ruth Slack (Ottawa, ON), and adenovirus expressing human cyclin D1 was obtained from Dr. J Albrecht (Minnesota) (24). Viruses were generated by ligation of either Lac Z or cyclin D1 into pACCMV.pLpA followed by cotransfection with pJM17 into 293 cells. Briefly, MCF-7 and ZR-75 cells were split and plated on coverslips. After attachment, cultures were infected with the Lac Z adenovirus or cyclin D1 adenovirus at a multiplicity of infection of 75 for 3 h in a minimum of medium without serum. Following infection, medium with 5% fetal bovine serum and 1 µM RA or vehicle was added to cultures for 96 h unless otherwise stated. For antisense Bcl-2 experiments, cells were transfected in serum-free medium at 37 °C/5% CO2 with the oligonucleotide 5'-TCTCCCAGCGTGCGCCAT-3' from Trilink Bio Technologies Inc. at a final concentration of 150 nM using Lipofection reagent (Invitrogen). A control oligonucleotide contained the same base composition in a random order. Four h later, the transfection medium was replaced with complete medium for the final 48 h of culture.

Mitochondrial Fractionation-- Subconfluent cultures were collected after trypsinization, centrifuged in medium containing 5% serum, and washed in phosphate-buffered saline (PBS) (137 mM NaCl, 27 mM KCl, 10 mM Na2HPO4, KH2PO4). The insoluble microsomal fraction containing mitochondria was isolated exactly as described (23) except that final supernatants were transferred to ultrafuge tubes and further clarified following a 1-h centrifugation at 100,000 × g.

Immunoblot-- Cultures were harvested into prechilled microfuge tubes in 500 µl of radioimmune precipitation assay buffer containing a protease inhibitor mixture. Lysates were processed, subjected to SDS-PAGE, and transferred to polyvinylidene difluoride membranes for immunodetection and visualization by chemiluminescence as described previously (23). Densitometric analysis was performed using a Kodak Image Station 440 CF.

Immunocytochemistry-- For Bax 6A7 staining, cells were cultured in 35-mm dishes on coverslips, and at the appropriate times, they were fixed with 3% formaldehyde in PBS at room temperature and then permeabilized for 2 min with 0.2% CHAPS in PBS. Mouse anti-Bax monoclonal 6A7 was used at a 1:300 dilution in 3% bovine serum albumin in PBS and incubated with coverslips for 1 h at room temperature. For double-staining, the anti-cyclin D1 polyclonal antibody was used at a dilution of 1:50. Immunostained cells were detected following a 1-h incubation at room temperature with CY3-labeled goat anti-mouse or fluorescein-labeled goat anti-rabbit secondary antibody. Cytochrome c staining (1:50) was performed on cold methanol-fixed cells, and antibody was detected with CY-3-conjugated donkey anti-rabbit secondary antibody. Coverslips were mounted with anti-fade (glycerol containing 1 mg/ml p-phenylene-diamine). For Hoechst staining, cells were fixed on coverslips with cold methanol at -20 °C for 5 min and then incubated in a 1:1500 dilution of Hoechst 33258 at room temperature, washed with PBS, and then mounted on coverslips with anti-fade. Intensely stained nuclei of reduced size were scored as apoptotic. Image capture and slide evaluations were performed using a Zeiss Axiophot fluorescence microscope equipped with Northern Eclipse image analysis software (EMPIX Imaging Inc., Mississauga, ON). All histogram bars represent results from evaluation of at least 1000 cells enumerated on coverslips from multiple microscopic fields.

Analysis of Caspase Activity-- Cells were trypsinized for 96 h following viral infection and culture in the presence or absence of RA, and 3 × 105 cells were resuspended in 300 µl of complete Dulbecco's modified Eagle's medium. 2 µl of propidium iodide solution (250 µg/ml) was added to the suspension just prior to flow analysis. Detection of caspase activity was performed using the CaspaTag fluorescein caspase VAD activity kit (Intergen, Purchase, NY) containing the inhibitor FAM-VAD-FMK according to the manufacturer's instructions. Caspase activity in camptothecin-treated cells was used as a positive control. Flow analysis of ungated fluorescence-labeled cells was performed on a Coulter Epics Altra cytometer (Hialeah, FL) equipped with an Argon laser and EXPO II software (Applied Cytometry Systems). Background levels of FAM-VAD-FMK and PI staining in uninfected cells were subtracted from experimental values.

Cell Cycle Analysis-- Cells were collected by centrifugation at 4 °C in PBS with 1 mM EDTA and then resuspended in 1 ml of ice-cold PBS/EDTA and fixed by the addition of 3 ml of 80% ice-cold ethanol. For flow cytometric analysis, an aliquot of cells was pelleted at 500 × g for 5 min, washed with PBS/EDTA, and then resuspended in PBS/EDTA containing 100 µg/ml RNase A for 20 min at room temperature and made 32 µM with propidium iodide. Following flow cytometry, data analysis was performed using the Multicycle AV program for Windows (Phoenix Flow Systems, San Diego, CA).

Assessment of Mitochondrial Membrane Potential-- For the last 2 h of culture, infected cells on coverslips that had been treated with 1 µM RA or vehicle for 96 h were incubated in medium containing 250 nM Mitotracker Red CM-H2XRos (Molecular Probes), which fluoresces only in cells with an intact mitochondrial membrane potential. Coverslips were then rinsed twice in PBS, fixed, and permeabilized as described above for immunostaining with the Bax 6A7 antibody and then stained with Hoechst.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Amplified Cyclin D1 Expression Results in a Conformational Change in Bax without Inducing Apoptosis-- To investigate the effects of amplified expression of cyclin D1, we infected MCF-7 and ZR-75 cells with an adenovirus expressing human cyclin D1 (adeno-cyclin D1) or the same virus expressing Lac Z (adeno-Lac Z). The immunoblot in Fig. 1A shows that cells infected with adeno-cyclin D1 express cyclin D1 at more than 20-fold (determined densitometrically) above the endogenous level assayed in adeno-Lac Z-infected cells at 96 h following infection. Staining for Lac Z in the adeno-Lac Z-infected cultures revealed Lac Z activity in at least 75% of the cells at 96 h after infection (not shown). Initially, we determined the sensitivity to RA-induced cell death and the availability of the N-terminal epitope of Bax in adeno-cyclin D1-expressing cells. Monoclonal antibody 6A7 (25) recognizes the N terminus of Bax associated with its mitochondrial membrane insertion and oligomerization early in apoptosis but does not bind to soluble Bax. Fig. 1B contains micrographs of Bax 6A7 immunoreactivity and Hoechst staining of MCF-7 cells infected with either adeno-Lac Z or cyclin D1. Numerous mAb 6A7-positive cells can be seen in the adeno-cyclin D1 cultures but not in the adeno-Lac Z-infected cultures. Strikingly, almost no apoptotic nuclei as determined morphologically following Hoechst stain were present in adeno-cyclin D1-infected cells despite the widespread Bax activation in these cultures. Fig. 1C shows that there were few 6A7-positive cells in adeno-Lac Z-infected MCF-7 and ZR-75 populations. RA increased the percentage 4-5-fold in both cell lines. In remarkable contrast, percentages of 6A7-positive cells in adeno-cyclin D1-infected MCF-7 and ZR-75 cells were increased 10- and 36-fold, respectively, over those observed in Lac Z virus-infected cells. Although RA further increased the percentage of 6A7-reactive cells, this increase was fractional as compared with the severalfold increase in apoptotic nuclei following RA treatment of adeno-cyclin D1-infected cells. Surprisingly, less than 2% of both untreated Lac Z and adeno-cyclin D1-infected MCF-7 and ZR-75 cells contained morphologically apoptotic nuclei. Only small increases in apoptotic nuclei were obtained after RA treatment of adeno-Lac Z-infected cultures. This contrasts with the much larger increase in percentages of apoptotic cells induced by RA in the cyclin D1 virus-infected cells. Thus cyclin D1 induces Bax translocation but not apoptosis in these breast cancer cell lines. Moreover, RA acts synergistically with cyclin D1 to increase apoptosis by a factor that far exceeds that by which it increases Bax translocation. When vehicle-treated adeno-cyclin D1-infected cells were left in culture past 96 h, what appeared morphologically to be necrotic cell death occurred involving cell swelling without blebbing beginning at about day 6 after infection.


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Fig. 1.   Amplified cyclin D1 expression results in N-terminal Bax epitope exposure in the absence of cell death. A, immunoblot analysis with cyclin D1 antibody of 15 µg of whole cell extracts from ZR-75 and MCF-7 cells infected with adenovirus expressing Lac Z or cyclin D1. As shown in B, MCF-7 cells grown on coverslips in the presence or absence of 1 µM RA for 96 h after infection with either adeno-Lac Z or adeno-cyclin D1 were immunostained with monoclonal antibody 6A7 or stained with Hoechst. Arrows indicate apoptotic nuclei as described under "Experimental Procedures." Bar, 100 µm. C, percentages of MCF-7 and ZR-75 cells immunoreactive with Bax 6A7 antibody and Hoechst-stained apoptotic nuclei. Graphs depict the mean of three separate experiments, and bars indicate standard errors which were 5% or less.

To ensure that cyclin D1 overexpression was coincident with activated Bax, we co-immunostained cells with both mAb 6A7 and anti-cyclin D1. Fig. 2A shows intense nuclear staining for cyclin D1 in cells indicated by the arrowheads. The accompanying panel shows that the same cells stain positively with mAb 6A7. A time course of cyclin D1 expression is shown in Fig. 2B, indicating that 24 h after viral infection, cyclin D1 protein levels began to increase and were markedly increased 96 h after adeno-cyclin D1 infection. A time course of the progressive increase in immunoreactivity with mAb 6A7 in Fig. 2C indicated that Bax activation began at levels of cyclin D1 expressed at ~72 h in both control and RA-treated cultures and increased further by 96 h. Together, these data indicate that high cyclin D1 expression results in a conformational change in Bax that does not itself cause apoptosis.


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Fig. 2.   Cyclin D1 expression correlates with Bax 6A7 immunostaining. As shown in A, adeno-cyclin D1-infected MCF-7 cells were fixed and co-immunostained with a polyclonal antibody against cyclin D1 and Bax 6A7. Secondary antibodies were conjugated to fluorescein isothiocyanate and CY3, respectively. Bar, 100 µm. B, Western blot analysis of cyclin D1 expression at the indicated times after adeno-cyclin D1 infection. C, time course of Bax 6A7 immunoreactivity in adeno-cyclin D1-infected cells. MCF-7 and ZR-75 cells grown on coverslips were fixed and immunostained with Bax 6A7 monoclonal antibody at the indicated times after infection with adeno-cyclin D1. Open bars, adeno-Lac Z-infected cultures; filled bars, adeno-cyclin D1-infected cultures. Percentages were derived from enumeration of at least 1000 cells.

Presence of Cytoplasmic Cytochrome c and Variable Loss of Mitochondrial Membrane Potential in Cyclin D1-overexpressing Cells-- The insertion of Bax into the mitochondrial membrane results in release of cytochrome c to the cytosol, potentially through Bax-associated pore formation (7, 26). To see whether the large increase in 6A7 immunostaining was associated with cytochrome c translocation, we treated MCF-7 cells with vehicle or RA for 96 h following infection with either the Lac Z or cyclin D1 virus and performed immunocytochemistry for cytochrome c. Fig. 3A shows punctate staining that was predominantly perinuclear in control Lac Z virus-infected cells. After RA treatment, about 5% of these cells showed evidence of more uniform staining throughout the cytoplasm, which often obscured the nucleus. By contrast, in control cyclin D1 virus-infected cultures, between 20 and 25% of the cells appeared to lose the perinuclear staining as immunoreactivity became more widespread throughout the cell, and this percentage appeared increased after the addition of RA. To more quantitatively compare the relative levels of cytochrome c release in these treatment groups, we next isolated membrane and cytosolic fractions from both RA-treated and untreated adeno-cyclin D1- and adeno-Lac Z-infected cultures for immunoblot analysis of cytochrome c. The results in Fig. 3B indicate that 96 h after infection, cytochrome c is clearly present in the cytoplasmic fractions from untreated adeno-cyclin D1-infected ZR-75 and MCF-7 cultures, and this level is slightly increased in RA-treated cultures. Immunoblots were reacted with anti-cytochrome c oxidase as a control for the integrity of the fractionation. Ratios of cytoplasmic:mitochondrial cytochrome c from two separate experiments each using MCF-7 and ZR-75 cells are shown below.


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Fig. 3.   Amplified cyclin D1 is associated with cytochrome c release and variable loss of membrane potential. As shown in A, Lac Z- or cyclin D1 virus-infected MCF-7 cells on coverslips were treated with vehicle or RA for 96 h and then fixed and stained with anti-cytochrome c as described under "Experimental Procedures." Arrows indicate cells with intact mitochondrial, predominantly perinuclear, punctate staining. Arrowheads indicate cells with cytoplasmic cytochrome c. See "Results" for details. As shown in B, 10 µg of soluble (S) and membrane fraction (M) from MCF-7 and ZR-75 cells infected with adeno-Lac Z or cyclin D1 virus were immunoblotted with anti-cytochrome c. Cytochrome c oxidase was used as a control for the integrity of the M fraction, and a cross-reactive band at 20 kDa was present only in the S fraction. Ratios derived from densitometric analysis of cytochrome c in membrane and cytosolic fractions from two separate experiments (expt 1 and expt 2) with each cell line are shown below. Both gels depicted are from expt 2. As shown in C, mitochondrial membrane potential in Bax 6A7-positive cells was detected using Mitotracker Red loading as described under "Experimental Procedures." MCF-7 cells infected with adeno-cyclin D1 were cultured for 96 h in the absence (upper control panels, CON) or presence (lower panel) of 1 µM RA. Cells were fixed and stained with Hoechst and immunostained with Bax 6A7. Arrows indicate Bax 6A7-immunopositive cells across the panels. Bar, 50 µm. D, quantization of cells positive for Bax 6A7 reactivity and Mitotracker Red fluorescence. Each bar represents the mean percentages derived from two separate experiments.

Depending on the cell type and the mode of induction of apoptosis, a number of studies have shown that the release of cytochrome c can precede or follow the loss of mitochondrial membrane potential. For example, although tumor necrosis factor-alpha treatment of hepatocytes results in simultaneous release of cytochrome c and mitochondrial membrane depolarization (27), UV- or staurosporine-treated HeLa and HL-60 cells do not lose mitochondrial membrane potential until well after the appearance of cytoplasmic cytochrome c (28, 29). To determine the status of mitochondrial membrane potential, we loaded cells for 1 h prior to harvest with the reduced form of MitoTracker Red and then immunostained with Bax 6A7 and counterstained with Hoechst. Fig. 3C shows bright punctate MitoTracker labeling in untreated MCF-7 cells infected with adeno-Lac Z. Adeno-cyclin D1-infected cultures also showed a similar pattern of MitoTracker labeling in many of the cells that also stained positive with the 6A7 antibody. Reduced staining was observed in the balance of 6A7-positive cells. Both these populations were devoid of apoptotic nuclei as determined by Hoechst staining. As expected, apoptotic nuclei were abundant in the RA-treated 6A7-positive adeno-cyclin D1-infected population, and a number of these cells showed a more diffuse pattern of MitoTracker staining or highly reduced staining. The percentages of mAb 6A7-positive cells displaying MitoTracker Red labeling are shown in Fig. 3D and indicate that over 50% of the untreated 6A7-positive cells retained mitochondrial membrane potential, whereas it was drastically reduced after RA treatment. Thus amplified expression of cyclin D1 causes widespread Bax activation and cytochrome c translocation, often without loss of mitochondrial membrane potential. Only after the addition of RA is there a more complete loss of membrane potential.

Cyclin D1 Overexpression Induces Bax Translocation but Does Not Alter Bax Protein Levels-- Overexpression of Bax can result in formation of Bax homodimers and mitochondrial membrane insertion; therefore, cyclin D1 overexpression could increase activated Bax by inducing an increase in Bax expression. The immunoblots in Fig. 4A indicate that after a 96-h infection, Bax or Bcl-2 levels were equivalent in Lac Z- and cyclin D1-expressing cell extracts. Concomitant treatment with RA resulted in either no change (MCF-7) or a slight increase (ZR-75) in Bax levels while producing a profound decrease in Bcl-2 levels, which we have seen previously in MCF-7 cells (23). Thus Bax activation in cyclin D1-infected cells is not the result of altered levels of Bax or Bcl-2. To assess Bax and Bcl-2 protein in mitochondria, the same membrane fractions used in the immunoblot in Fig. 3A were subjected to analysis for Bax and Bcl-2. Fig. 4B shows that mitochondrial Bax levels are more than doubled in cyclin D1-expressing cells as compared with those expressing Lac Z. The blot was reincubated with Bcl-2 antibody and, as expected, cyclin D1 had no effect on mitochondrial Bcl-2 levels. Importantly, although RA decreased mitochondrial Bcl-2 in the cyclin D1-expressing cultures, it had no effect on Bax levels. Mitochondrial heat shock protein (mt hsp) 70 is exclusively localized to mitochondria in most cells (30) and was used as a marker for intact mitochondria and a gel-loading control. These data support the notion that RA-induced apoptosis in cells ectopically expressing cyclin D1 is not the result of increased mitochondrial Bax but rather is due to a reduction in Bcl-2 levels.


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Fig. 4.   Bax translocates to mitochondria, but levels are unchanged in cyclin D1 virus-infected cultures. A, immunoblot analysis of Bax and Bcl-2 in cell extracts from vehicle and RA-treated Lac Z or cyclin D1-overexpressing cells. Actin was used as a control for gel loading. As shown in B, membrane fractions from Lac Z- and cyclin D1 virus-infected cells were subjected to immunoblot with a polyclonal anti-Bax and anti-Bcl-2. Subcellular fraction integrity and loading was confirmed using anti-proliferating cell nuclear antigen and anti-mt hsp 70 as markers for nuclear/cytoplasmic contamination and mitochondria, respectively.

Ectopic Cyclin D1 Expression Induces p53 and G2/M Phase Arrest-- p53 can mediate both G1 and G2 arrest as well as apoptosis. G1 arrest is mostly attributed to p21 induction, whereas arrest in G2 in response to damaged DNA or nucleotide depletion in S phase involves induction of Gadd45, p21, and 14-3-3F, all of which contribute to cdc2 inhibition (31). Induction of apoptosis is facilitated by a number of p53 targets including Bax, Noxa, PUMA, as well as components of the death receptor pathway (32). To determine whether p53 was induced by cyclin D1 overexpression and might therefore be involved in the response to cyclin D1 overexpression, we performed immunoblot analysis of whole cell extracts from adeno-cyclin D1 and Lac Z-infected MCF-7 and ZR-75 cells in the presence or absence of RA. The results in Fig. 5A show that p53 protein levels were induced in both cell lines by cyclin D1; however, RA did not augment these levels. Although MCF-7 reportedly contains wild-type p53, it remained possible that some highly passaged isolates may have acquired p53 mutation. Fig. 5B shows that adeno-cyclin D1 infection increased p53 expression by 24 h, and it remained expressed throughout the duration of the experiment. Transcriptionally competent p53 regulates the expression of the oncoprotein, mdm2, which in turn facilitates ubiquitin-mediated degradation of p53 (reviewed in Ref. 32). Incubation of the same blot in mdm2 antibody shows that mdm2 expression after a short latency period increased in parallel with that of p53. Thus p53 protein produced in MCF-7 cells is transcriptionally active. We also noticed that cells became enlarged to nearly twice their normal size, suggestive of a potential premitotic arrest in G2/M. To determine whether cyclin D1 levels mediated differential effects on the cell cycle, we performed cell cycle analysis on adeno-cyclin D1-infected MCF-7 cells at 24, 48, and 96 h after infection. Fig. 5C shows that at 24 h after infection, cultures contained elevated percentages of S phase cells as compared with Lac Z controls. However, by 48 h, more than half of cyclin D1 cells were in G2/M as compared with only 20% of control cells. At 72 h, two-thirds of the cyclin D1-expressing cells were in G2/M as compared with less than 30% of Lac Z control cells. Although RA usually produces a G1 accumulation in breast cancer cells (33), amplified cyclin D1 expression clearly superceded its effect. Thus cyclin D1 induces p53 expression associated with both Bax translocation and G2/M arrest.


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Fig. 5.   Induction of p53 and G2 block following cyclin D1 virus infection. As shown in A, MCF-7 and ZR-75 cells infected with the Lac Z or cyclin D1 virus were cultured in the presence or absence of 1 µM RA for 96 h as described under "Experimental Procedures." Whole cell extracts were immunoblotted for p53 and actin. As shown in B, MCF-7 cell extracts were obtained at the indicated times after cyclin D1 virus infection and immunoblotted with anti-p53, anti-mdm2, and anti-actin. The Lac Z virus-infected cell extracts were obtained 96 h after infection. C, cell cycle analysis of Lac Z- and cyclin D1-infected MCF-7 cells. Cell cycle analysis was performed as described under "Experimental Procedures" at the indicated times after viral infection.

Activation of Bax Following Cyclin D1 Overexpression Is p53-dependent-- To assess whether or not p53 played a key role in the activation of Bax adeno-cyclin D1 infection, we stably expressed HPV-E6 in MCF-7 cells and assayed Bax 6A7 reactivity. Fig. 6A shows that the HPV-E6 protein significantly reduced the level of p53 protein in the stably transfected MCF-7 cells (clones 3 and 9). Moreover, Fig. 6B indicates that this expression was accompanied by a drastic reduction in Bax 6A7-positive cells as compared with untransfected control cells after cyclin D1 expression. Whereas RA induced 6A7 reactivity in between 40 and 50% of cyclin D1-infected MCF-7 cells, less than 20% of MCF-7(HPV-E6) cells were 6A7 immuno-positive. Thus p53 participates in signaling the Bax conformational change following cyclin D1 overexpression. Although p53 was clearly required for Bax activation, it was not possible to differentiate between p53-dependent activation of Bax and a putative role for p53 in mitochondrial cytochrome c release during cyclin D1 overexpression.


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Fig. 6.   Cyclin D1-induced Bax activation is p53-dependent. As shown in A, MCF-7 cells were transfected with an HPV-16 E6 expression plasmid. The resulting levels of p53 assessed by immunoblot of whole cell extracts from untransfected cells (con) and two clones (3 and 9) are shown. B, evaluation of mAb 6A7-positive cells and apoptotic nuclei following Lac Z or cyclin D1 virus infection and culture in the presence or absence of 1 µM RA. Graphs represent the mean of percentages derived from three separate experiments, and bars indicate standard errors which were 6% or less.

Bcl-2 Prevents Cyclin D1-induced Bax Activation but Not Cell Cycle Arrest of Cyclin D1 Adenovirus-infected Cells-- Bcl-2 overexpression can prevent many types of cell death and has been shown to prevent conformational changes in Bax associated with Fas-induced cell death (34). Since cell death and activation of Bax was exacerbated in adeno-cyclin D1-infected MCF-7 cells following RA treatment, and RA decreases Bcl-2 levels in MCF-7 cells (23), we investigated whether constitutive expression of Bcl-2 alone was sufficient to prevent Bax activation and death in RA-treated cyclin D1-expressing MCF-7 cells. Using previously characterized clones of MCF-7(Bcl-2) clones (35), we infected pooled populations of clones with adeno-cyclin D1 or adeno-Lac Z and determined the effects of Bcl-2 overexpression on Bax conformational change. Fig. 7A shows that, in contrast to untransfected MCF-7 cells, activated Bax was detected at very low levels in untreated adeno-cyclin D1-infected cultures. Even when MCF-7(Bcl-2) cells were cultured in RA for 96 h, Bcl-2 strongly attenuated the increase in Bax N-terminal exposure to the extent that the percentages were more than 10-fold less than those observed in cyclin D1 virus-infected control MCF-7 cells. Cell death was also reduced substantially by Bcl-2 as determined by Hoechst staining, especially in RA-treated cultures of adeno-cyclin D1-infected MCF-7(Bcl-2) cells. Therefore, in adeno-cyclin D1-infected cells, overexpressed but clearly not endogenous levels of Bcl-2 can prevent Bax N-terminal exposure in the absence or presence of RA.


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Fig. 7.   Bcl-2 prevents cyclin D1-induced Bax activation and RA-induced cell death. As shown in A, three pooled MCF-7 cell lines stably expressing Bcl-2 (Teixeira et al. (35)) were infected with adeno-cyclin D1 or adeno-Lac Z and then cultured in the presence or absence of 1 µM RA. Cells were stained with Hoechst and immunostained with the Bax 6A7 monoclonal antibody as described under "Experimental Procedures." The graphs show the results of enumeration of the percentages of mAb 6A7-positive cells and apoptotic nuclei in adeno-Lac Z and adeno-cyclin D1-infected cells. Graphs represent mean percentages from two separate experiments. Con, untransfected control cells. B, cell cycle analysis of MCF-7(Bcl-2) clones. Cell cycle analysis was performed as described under "Experimental Procedures" at the indicated times following adeno-cyclin D1 infection of three pooled Bcl-2- expressing clones.

We next asked whether the inhibition of Bax activation by Bcl-2 also prevented the G2/M block induced by cyclin D1. Results of cell cycle analysis in Fig. 7B indicated that, similar to untransfected MCF-7 cells, adeno-cyclin D1 infection of MCF-7(Bcl-2) cells still induced a G2/M block, thus indicating that the cell cycle effects were dissociable from the activation of Bax.

Cyclin D1 Fails to Induce Caspase Activity in the Absence of RA or Antisense Bcl-2-- Release of cytochrome c from mitochondria constitutes one of the terminal steps in the activation of caspase-9 within the apoptosome. However, release of cytochrome c does not necessarily result in caspase activation since IAP molecules are capable of inhibiting the activity of caspase-9 even in the presence of cytochrome c and ATP. The absence of high levels of cell death in the adeno-cyclin D1-infected cells suggested that release of cytochrome c in these cultures was insufficient to activate apoptosis. To assess terminal apoptotic events, we measured caspase activity using a fluorescence-tagged FAM-VAD-FMK inhibitor that binds irreversibly to all active caspases except caspase-4 and -10 in MCF-7 and ZR-75 cells infected with either adeno-Lac Z or cyclin D1 virus. Table I displays the results of flow cytometric analysis of fluorescent cells 96 h after infection in the presence or absence of RA. Concomitant propidium iodide staining allowed evaluation of cells at various stages of death including those that were only VAD-positive (early apoptosis), both VAD and PI-positive (mid-apoptosis), and only PI-positive (late apoptosis). Caspase activity was clearly present in both cell lines infected with Lac Z, although these levels were ~15% higher in MCF-7 cells as compared with ZR-75 cells, demonstrating a higher level of adenoviral toxicity in MCF-7 cells. The addition of RA to Lac Z-infected cells only weakly reduced the percentage of healthy cells. Importantly, in neither cell line did the expression of cyclin D1 alone result in an increase in VAD-positive cells over Lac Z controls. Only in the presence of RA was there a considerable increase in the percentage of VAD-positive cells from either cell line. Most of these cells were present in the VAD/PI-positive population, suggesting that apoptosis was not acute at 96 h. Since a clear proapoptotic effect of RA is the reduction of Bcl-2 levels, we asked whether antisense Bcl-2-mediated down-regulation of Bcl-2 would produce a similar effect on caspase activation. Fig. 8A shows that transfection of the oligonucleotide resulted in a greater than 50% decrease in Bcl-2 levels. This effect persisted for at least 48 h after transfection (not shown); therefore, transfection of the oligonucleotides into virus-infected cells was for the last 48 h of culture. Transfection of control oligonucleotide into cyclin D1-infected cells resulted in nearly 40% 6A7 mAb positivity (Fig. 8B), whereas antisense Bcl-2 increased this percentage to 55%. Thus the effect of reduction of Bcl-2 protein levels using antisense Bcl-2 closely paralleled the effects of the addition of RA on Bax activation. Importantly, 6A7-positive cells increased following antisense Bcl-2 transfection into Lac Z-infected cells considerably more than after RA treatment, indicating a direct effect of the Bcl-2 antisense on Bax activation. To assess the associated induction of caspase activity, we performed flow cytometry on CaspaTag-labeled Bcl-2 antisense-treated cells. The result of this analysis in the lower panel of Table I indicates that, similar to RA, antisense Bcl-2 increased the percentage of cells containing active caspase. Moreover, these values are likely an underestimate of VAD- and/or PI-positive cells, based on the fact that many cells were floating or not intact by the end of the study. Thus, on one level, Bcl-2 controls the activation of Bax following the cyclin D1-induced death signal, and on another level, it acts as a determinate of caspase activation.


                              
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Table I
Cyclin D1-induced caspase activity and cell death


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Fig. 8.   Bcl-2 antisense reduces Bcl-2 and augments the percentage of mAb 6A7-positive cells. A, immunoblot analysis of Bcl-2 expression 48 h after transfection of MCF-7 cells with either control (con) oligonucleotide or antisense Bcl-2. Actin reactivity was used as an internal gel loading control. As shown in B, following infection with either Lac Z or cyclin D1 virus and transfection of either control or Bcl-2 antisense oligonucleotide for the final 48 h of culture, cells on coverslips were immunostained with mAb 6A7. Graphs represent mean percentages of positive cells from three separate experiments, and bars indicate standard errors, which were 8% or less.

Ectopic Cyclin D1 Induces Smac Release from Mitochondria-- One reason for the lack of caspase induction following cytochrome c release in cells ectopically expressing cyclin D1 might be either a lack of or a weak release of the Smac/DIABLO protein from the mitochondrial compartment. This is reasonable since Smac/DIABLO is thought to be contained in submitochondrial locations separate from cytochrome c (36). To assay Smac release, we subjected mitochondrial and cytoplasmic extracts to immunoblotting with an anti-Smac/DIABLO antibody. Using the same extracts as in Fig. 3, Fig. 9A shows that Smac protein is found predominately in the membrane fraction of Lac Z virus-infected cells in the presence or absence of RA, although some is also present in the cytoplasm, likely as a result of low level viral toxicity. The ratio of membrane to soluble Smac decreased by half after ectopic cyclin D1 expression and even further when cells were treated with RA. Given the effect of RA on Bcl-2 levels, we predicted that antisense Bcl-2 oligonucleotide transfection into cyclin D1 virus-infected cells would achieve a similar release of Smac as did RA. The results in Fig. 9B indicate that although control oligonucleotide transfection of cyclin D1-expressing cells caused Smac release to a similar extent as in untransfected cells, transfection of Bcl-2 antisense caused a profound induction of Smac release comparable with that after RA treatment of control oligonucleotide-transfected cells, which was further augmented by RA. Notably, although mt hsp 70 was retained almost entirely in the membrane fraction after RA treatment of control oligonucleotide-transfected cells ectopically expressing cyclin D1, release or leakage of this protein from mitochondria occurred after Bcl-2 antisense treatment, perhaps reflecting a complete loss of mitochondrial membrane integrity in some cells as a result of radical Bcl-2 protein reduction. Thus, although adeno-cyclin D1 infection causes Smac release, it is insufficient to induce caspase activation in the absence of a decrease in Bcl-2 protein levels.


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Fig. 9.   Amplified cyclin D1 expression induces mitochondrial Smac release. As shown in A, membrane (M) and soluble (S) fractions used in Fig. 3 from adeno-cyclin D1- and adeno-Lac Z-infected cells treated with vehicle or 1 µM RA were subjected to immunoblot with anti-Smac. The ratios of membrane to soluble Smac were determined from densitometric quantization of Smac levels in the two fractions. As shown in B, adeno-cyclin D1-infected cells were transfected with either control or Bcl-2 antisense oligonucleotide and membrane and soluble fractions isolated as in panel A. Immunoblot analysis was performed with antibodies to Smac and mt hsp 70 as a control for mitochondrial integrity. The Smac M/S ratio was determined as in panel A. Blots are representative of two independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Most proapoptotic agents or stimuli do not simply result in formation of Bax dimers but also intersect with other facets of the death pathway, resulting in the activation of a complete apoptotic program. This has made it difficult to evaluate the relative importance of individual factors in the mitochondrial death pathway without ectopic expression or overexpression of either engineered or wild-type Bcl-2 family members. Rossé et al. (37) showed that overexpression of transiently transfected Bax resulted in its mitochondrial translocation, cytochrome c release, caspase activation, and cell death. Although Bax-induced cytochrome c release was not prevented by simultaneous overexpression of Bcl-2 in that study, the activation of caspase-3 was inhibited. Taken together with our data, this suggests that the ratio of Bcl-2 to Bax required to prevent cytochrome c release must be higher than that needed to prevent caspase activation regardless of the mode of activation of cytochrome c release. Thus the post-mitochondrial steps in the death pathway are more difficult to achieve than the initiating events for a given level of Bcl-2.

The working hypothesis for regulation of the mitochondrial cell death pathway is that the ratio between proapoptotic molecules such as Bax and Bak and the antiapoptotic molecules, Bcl-2 and Bcl-xL, helps to determine the susceptibility of cells to die in response to a death signal (38). RA/antisense-mediated decreases in Bcl-2 expression could contribute to the total number of cells in which cyclin D1-induced Bax translocation occurs or could itself induce Bax activation in those cells either infected with low numbers or not infected with the cyclin D1 virus. However, neither agent was synergistic with cyclin D1 in terms of the activation of Bax. Most importantly, the fold increases in caspase activity and apoptosis after RA/Bcl-2 antisense treatment of cyclin D1-infected cells were dramatically higher than in untreated cells and in RA-treated Lac Z-infected cultures. This result demonstrates pathway progression from the mitochondrial membrane to caspase activation and death within the time frame of the study primarily as a result of decreased Bcl-2.

Cyclin D1 is a key determinant in some forms of tumorigenesis, yet similar to other proliferative protooncogenes such as c-Myc (39), increased expression can also induce cell death. The series of experiments presented herein demonstrates for the first time the cellular response to high level expression of cyclin D1. Table II summarizes the effects of cyclin D1 on Bax N-terminal exposure and caspase/death induction in breast cancer cells, clearly illustrating that increased expression of cyclin D1 initiates apoptotic events up to but not including activation of caspases and death. Thus Bax translocation and cytochrome c release are insufficient for caspase activation. One reason may be that endogenous levels of Bcl-2 are sufficiently high to prevent this activation. To this end, it has been postulated that Bcl-2 may act at a post-mitochondrial step to inhibit Apaf-1 activation of caspase-3, although the most recent evidence suggests that they do not (40 and references therein). Bcl-2 can also inhibit Bax-induced cytoplasmic acidification, which would normally facilitate caspase activation (41) and may do so by regulating mitochondrial proton flux (42). A failure to release the IAP inhibitor Smac/DIABLO from the mitochondria might also provide a mechanism for lack of caspase activation. However, in contrast to caspase activation, the release of mitochondrial Smac/DIABLO appeared to reflect the level of Bax activation and cytochrome c released in adeno-cyclin D1-infected cells. Although caspase inhibitors can prevent the release of Smac/DIABLO (36), there may be sufficient basal activity in cells to allow for its release. Consistent with this, both RA and Bcl-2 antisense caused increased Smac release in the present study.


                              
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Table II
Comparative analysis of cyclin D1-induced Bax and caspase activation

Why overt apoptosis does not occur despite cytochrome c release remains in question, although Martinou et al. (9) have invoked scenarios wherein there remains sufficient cytochrome c associated with the electron transport chain complexes or reassociation of free cytochrome c to maintain respiration and polarization. Where caspase activation has been blocked (43, 44), cells can, in fact, recover from complete cytochrome c translocation. There is good evidence that even the loss of mitochondrial membrane potential is reversible and not a final commitment to cell death (45). In the case of Bid- or Bax-mediated cytochrome c release, only the outer mitochondrial membrane is permeable, whereas the inner membrane remains intact (46), an observation which corroborates our finding that MitoTracker staining remained intact in many of the Bax 6A7-positive cells. Cytochrome c release in individual cells is rapid and complete and independent of caspase activation (47). This is an important point, given that we did not detect a significant increase in caspase activity in adeno-cyclin D1-infected cells without the addition of RA or Bcl-2 antisense treatment.

Our data show that p53 is pivotal to Bax activation following cyclin D1 overexpression. There is growing evidence that p53 may act at the level of the mitochondria to promote cell death in a non-transcriptional mode (48, 49). From the transcriptional standpoint, a number of p53-responsive genes are thought to contribute to p53-mediated apoptosis including bax, bak, NOXA, and PUMA (32, 50). NOXA and PUMA may act in a Bid-like capacity to facilitate opening of the Bax molecule. Thus, although we did not observe any increase in Bax after cyclin D1 overexpression, it is conceivable that p53-mediated induction of the latter genes plays a role in the observed Bax activation. The lack of Bax activation in HPV-16 E6-expressing cells and the fact that cyclin D1 overexpression did not produce Bax activation in several breast cancer cell lines lacking functional p53 2 indicate that cyclin D1-induced Bax activation is p53-dependent. It is important to note that HPV-18 E6 can also induce degradation of Bak, thereby inhibiting apoptosis in a p53-independent manner (51). However, immunoblot analysis of Bak in MCF-7(HPV-18 E6) cells showed only a marginal decrease in Bak expression, and Bak was not present in the mitochondrial fraction before or after cyclin D1 overexpression (data not shown).

Evidently the level of cyclin D1 expression determines the cellular response. Normally, cyclin D1 is required for G1 progression and is decreased in subsequent phases of the cell cycle (1, 2). Two groups have shown that constitutive cyclin D1 expression sensitizes cells to serum starvation-induced death (3, 52). We have found that MCF-7 cells constitutively expressing cyclin D1 were sensitized to RA (23). Pagano et al. (52) showed that acute cyclin D1 overexpression prevented normal fibroblasts from entering S phase, an effect that was prevented by coexpressed proliferating cell nuclear antigen. Thus, although cyclin D1:proliferating cell nuclear antigen associations form part of the G1 checkpoint in normal cells, at least in breast cancer cells, progression into S phase is clearly permitted since the block is now in the G2 phase. The mechanism of cyclin D1-induced Bax translocation remains unclear. Levels of cyclin D1 are normally reduced after G1 as a result of glycogen synthase kinase-3beta phosphorylation and subsequent targeting for degradation or nuclear export (53). The high cyclin D1 levels in the infected MCF-7 cells may have saturated the glycogen synthase kinase-3beta enzyme and/or the nuclear exporter, resulting in constitutive nuclear cyclin D1. Alt et al. (53) have speculated that nuclear cdk4-cyclin D1 complexes may phosphorylate proteins in S phase that are normally substrates for cdk2, resulting in perturbation of the timing of activation of the DNA synthesis machinery. At moderate levels of cyclin D1, this may result in gene amplification events. On the other hand, higher levels could induce sufficient genetic instability to result in activation of the DNA damage checkpoint and subsequent induction of p53. Thus cyclin D1-initiated apoptosis provides the first example of a death signal, which demonstrates that Bcl-2 can function at different levels to regulate both mitochondrial and post-mitochondrial events.

    FOOTNOTES

* This work supported in part by Grant 98-B062 from The American Institute for Cancer Research (to M. A. C. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, ON, Canada K1H 8M5. Tel.: 613-562-5800 (ext. 8366); Fax: 613-562-5636; E-mail: cpratt@uottawa.ca.

Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M209650200

2 M. A. C. Pratt and M.-Y. Niu, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: CDK, cyclin-dependent kinase; RA, retinoic acid; HPV, human papillomavirus; IAP, inhibitor of apoptosis; mAb, monoclonal antibody; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; mt hsp, mitochondrial heat shock protein; PI, propidium iodide; FAM-VAD-FMK, carboxyfluorescein analog of benzyloxycarbonylvalylalanyl aspartic acid fluoromethyl ketone.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Hunter, T., and Pines, J. (1994) Cell 79, 573-582[Medline] [Order article via Infotrieve]
2. Sherr, C. J. (1996) Science 274, 1672-1677[Abstract/Free Full Text]
3. Han, E. K.-H., Begemann, M., Sgambato, A., Soh, J.-W., Doki, Y., Xing, W.-Q., Liu, W., and Weinstein, I. B. (1996) Cell Growth & Differ. 7, 699-710[Abstract]
4. Sofer-Levi, Y., and Resnitzky, D. (1996) Oncogene 13, 2431-2437[Medline] [Order article via Infotrieve]
5. Katayama, K., Dobashi, Y., Kitagawa, M., Kamekura, S., Kawai, M., Kadoya, Y., and Kameya, T. (2001) FEBS Lett. 509, 382-388[CrossRef][Medline] [Order article via Infotrieve]
6. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489[Medline] [Order article via Infotrieve]
7. Gross, A., McDonnell, J. M., and Korsmeyer, S. J. (1999) Genes Dev. 18, 1899-1911
8. Reed, J. C. (1998) Oncogene 17, 3225-3236[CrossRef][Medline] [Order article via Infotrieve]
9. Martinou, J. C., Desagher, S., and Antonsson, B. (2000) Nat. Cell Biol. 2, E41-E43[CrossRef][Medline] [Order article via Infotrieve]
10. Eskes, R., Desagher, S., Antonsson, B., and Martinou, J.-C. (2000) Mol. Cell. Biol. 20, 929-935[Abstract/Free Full Text]
11. Nechushtan, A., Smith, C. L., Hsu, Y.-T., and Youle, R. J. (1999) EMBO J. 18, 2330-2341[Abstract/Free Full Text]
12. Wolter, K. G., Hsu, Y. T., Smith, C. L., Nechushtan, A, Xi, X. G., and Youle, R. J. (1997) J. Cell Biol. 139, 1281-1292[Abstract/Free Full Text]
13. Gross, A., Jockel, J., Wei, M. C., and Korsmeyer, S. J. (1998) EMBO J. 17, 3878-3885[Abstract/Free Full Text]
14. Huang, D. C. S., and Strasser, S. (2000) Cell 103, 839-842[Medline] [Order article via Infotrieve]
15. Cheng, E. H.-Y. A., Wei, M. C., Weiler, S., Flavell, R. A., Mak, T. W., Lindsten, T., and Korsmeyer, S. J. (2001) Mol. Cell 8, 705-711[CrossRef][Medline] [Order article via Infotrieve]
16. Slee, E., Harte, M. T., Kluck, R. M., Wolf, B. B., Casiano, C. A., Newmeyer, D. D., Wang, H.-G., Reed, J. C., Nicholson, D. W., Alnemri, E. S., Green, D. R., and Martin, S. J. (1999) J. Cell Biol. 144, 281-292[Abstract/Free Full Text]
17. Deveraux, Q. L., and Reed, J. C. (1999) Genes Dev. 13, 239-252[Free Full Text]
18. Miller, L. K. (1999) Trends Cell Biol. 9, 323-328[CrossRef][Medline] [Order article via Infotrieve]
19. Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Cell 102, 33-42[Medline] [Order article via Infotrieve]
20. Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G. E., Moritz, R. L., Simpson, R. J., and Vaux, D. L. (2000) Cell 102, 43-53[Medline] [Order article via Infotrieve]
21. Borner, C., and Monney, L. (1999) Cell Death Differ. 6, 497-507[CrossRef][Medline] [Order article via Infotrieve]
22. Green, D. R., and Reed, J. C. (1998) Science 281, 1309-1312[Abstract/Free Full Text]
23. Niu, M.-Y., Menard, M., Reed, J. C., Krajewski, S., and Pratt, M. A. C. (2001) Oncogene 20, 3506-3518[CrossRef][Medline] [Order article via Infotrieve]
24. Hansen, L. K., and Albrecht, J. H. (1999) J. Cell Sci. 112, 2971-2981[Abstract/Free Full Text]
25. Hsu, Y.-T., and Youle, R. J. (1998) J. Biol. Chem. 273, 10777-10783[Abstract/Free Full Text]
26. Desagher, S., and Martinou, J.-C. (2000) Trends Cell Biol. 10, 369-377[CrossRef][Medline] [Order article via Infotrieve]
27. Bradham, C. A., Qian, T., Streetz, K., Trautwein, C., Brenner, D. A., and Lemasters, J. J. (1998) Mol. Cell. Biol. 18, 6353-6364[Abstract/Free Full Text]
28. Bossy-Wetzel, E., Newmeyer, D. D., and Green, D. R. (1998) EMBO J. 17, 37-49[Abstract/Free Full Text]
29. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T. I., Jones, D. P., and Wang, X. (1997) Science 275, 1129-1132[Abstract/Free Full Text]
30. Dahlseid, J. N., Lill, R., Green, J. M., Xu, X., Qui, Y., and Pierce, S. K. (1994) Mol. Biol. Cell 5, 1265-1275[Abstract]
31. Taylor, W. R., and Stark, G. R. (2001) Oncogene 20, 1803-1815[CrossRef][Medline] [Order article via Infotrieve]
32. Balint, E., and Vousden, K. H. (2001) Br. J. Cancer 85, 1813-1823[Medline] [Order article via Infotrieve]
33. Teixeira, C., and Pratt, M. A. C. (1997) Mol. Endocrinol. 11, 1191-1202[Abstract/Free Full Text]
34. Murphy, K. M., Streips, U. N., and Lock, R. B. (2000) J. Biol. Chem. 275, 17225-17228[Abstract/Free Full Text]
35. Teixeira, C., Reed, J. C., and Pratt, M. A. C. (1995) Cancer Res. 55, 3902-3907[Abstract]
36. Adrain, C., Creagh, E. M., and Martin, S. J. (2001) EMBO J. 20, 6627-6636[Abstract/Free Full Text]
37. Rossé, T., Olivier, R., Monney, L., Rager, M., Conus, S., Fellay, I., Jansen, B., and Borner, C. (1998) Nature 391, 496-499[CrossRef][Medline] [Order article via Infotrieve]
38. Oltvai, Z. N., Milliman, V., and Korsmeyer, S. J. (1993) Cell 74, 609-619[Medline] [Order article via Infotrieve]
39. Hueber, A. O., and Evan, G. I. (1998) Trends Genet. 14, 364-367[CrossRef][Medline] [Order article via Infotrieve]
40. Conus, S., Rossé, T., and Borner, C. (2000) Cell Death Differ. 7, 947-954[CrossRef][Medline] [Order article via Infotrieve]
41. Matsuyama, S., Llopis, J., Devereaux, Q. L., Tsien, R. Y., and Reed, J. C. (2000) Nat. Cell Biol. 2, 318-325[CrossRef][Medline] [Order article via Infotrieve]
42. Shimizu, S., Eguchi, Y., Kamike, W., Funahashi, Y., Mignon, A., Lacronique, V., Matsuda, H., and Tsujimoto, Y. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1455-1459[Abstract/Free Full Text]
43. Deshmukh, M., Kuida, K., and Johnson, E. M. (2000) J. Cell Biol. 150, 131-143[Abstract/Free Full Text]
44. von Ahsen, O., Renken, C., Perkins, G., Kluck, R. M., Bossy-Wetzel, E., and Newmeyer, D. (2000) J. Cell Biol. 150, 1027-1036[Abstract/Free Full Text]
45. Minamikawa, T., Williams, D. A., Bowser, D. N., and Nagley, P. (1999) Exp. Cell Res. 246, 26-37[CrossRef][Medline] [Order article via Infotrieve]
46. Shimizu, S., and Tsujimoto, Y. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 577-582[Abstract/Free Full Text]
47. Goldstein, J. C., Waterhouse, N. J., Juin, P., Evan, G. I., and Green, D. R. (2000) Nat. Cell Biol. 2, 156-162[CrossRef][Medline] [Order article via Infotrieve]
48. Marchenko, N. D., Zaika, A., and Moll, U. M. (2000) J. Biol. Chem. 275, 16202-16212[Abstract/Free Full Text]
49. Schuler, M., Bossy-Wetzel, E., Goldstein, J. C., Fitzgerald, P., and Green, D. R. (2000) J. Biol. Chem. 275, 7337-7342[Abstract/Free Full Text]
50. Miyashita, T., and Reed, J. C. (1995) Cell 80, 293-299[Medline] [Order article via Infotrieve]
51. Thomas, M., and Banks, L. (1998) Oncogene 27, 2943-2954[CrossRef]
52. Pagano, M., Theodoras, A. M., Tam, S. W., and Draetta, G. F. (1994) Genes Dev. 8, 1627-1639[Abstract]
53. Alt, J. R., Cleveland, J. L., Hannink, M., and Diehl, J. A. (2000) Genes Dev. 14, 3102-3114[Abstract/Free Full Text]


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