lum, and nuclear envelope. Bcl-2 is a member of a family of cell death regulators and functions to inhibit apoptosis. Using confocal microscopy and immunoblot-.
Oncogene (1997) 15, 2767 ± 2772 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
Bcl-2 inhibits p53 nuclear import following DNA damage Alexander Beham*, Maria C Marin*, Antonio Fernandez, John Herrmann, Shawn Brisbay, Ana M Tari1, Gabriel Lopez-Berestein1, Guillermina Lozano2, Mona Sarkiss and Timothy J McDonnell Departments of Molecular Pathology, 1Immunobiology and Drug Carriers and 2Molecular Genetics, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Blvd-Box 89, Houston, Texas 77030, USA
Bcl-2 is an integral membrane oncoprotein that localizes to membranes of the mitochondria, endoplasmic reticulum, and nuclear envelope. Bcl-2 is a member of a family of cell death regulators and functions to inhibit apoptosis. Using confocal microscopy and immunoblotting we show that the ability of bcl-2 to suppress cell death following genotoxic damage can be a consequence of inhibiting nuclear import of induced wild-type p53 protein. Our data suggests that the ability of bcl-2 to modulate tracking events is not cell type speci®c. These data support a `gatekeeper' mechanism for cell death suppression by bcl-2. Keywords: apoptosis; bcl-2; nuclear import; p53; prostate cancer
Introduction Bcl-2 has been demonstrated to signi®cantly suppress, or delay, the induction of cell death in a variety of experimental systems, and the deregulated expression of bcl-2 has been shown to directly contribute to multistep carcinogenesis in vivo (McDonnell and Korsmeyer, 1991). It has been proposed that bcl-2 function may be related to its ability to inhibit cellular damage resulting from free radicals (Hockenbery et al., 1993) and sustained increases in intracellular Ca2+ (Lam et al., 1994; Marin et al., 1996) associated with cell death induction. Interaction of bcl-2 with other bcl-2 family member proteins, such as bax, has also been shown to modulate bcl-2 function (Oltvai et al., 1993). The wild-type p53 tumor suppressor gene is able to mediate a cell cycle checkpoint and apoptotic cell death induction (Kastan et al., 1992; Yonish-Rouach et al., 1991). Induction of apoptosis by p53 is thought to play a critical role in the elimination of cells following DNA damage (Lowe et al., 1993a; Clarke et al., 1993). In addition, inactivating mutations of p53, similar to overexpression of bcl-2, confer resistance to cell death induction by radiation and multiple chemotherapeutic agents (Miyashita and Reed, 1992; Lowe et al., 1993b). Interestingly, the presence of p53 mutations has been shown to inversely correlate with bcl-2 expression in various tumor types including breast and prostate cancer (Silvestrini et al., 1994; McDonnell et al., 1997). One implication of these observations is that, with
Correspondence: TJ McDonnell *AB and MCM contributed equally to this manuscript Received 12 July 1997; revised 28 July 1997; accepted 29 July 1997
respect to the regulation of apoptosis, p53 and bcl-2 may serve as an eector and repressor of a common cell death pathway. Although bcl-2 is able to inhibit p53-dependent apoptosis induction following genotoxic damage (Chiou et al., 1994; Marin et al., 1994), the mechanistic basis of this inhibition has not been elucidated. Using immunoblotting procedures and scanning laser confocal microscopy we demonstrate that bcl-2 confers resistance to cell death induction in prostate carcinoma cells possessing wild-type p53 treated with girradiation. Although p53 protein was induced to equivalent levels in the bcl-2 expressing and control clones, the nuclear import of p53 protein following DNA damage was signi®cantly inhibited only in the former. Furthermore, enforced expression of bcl-2 signi®cantly inhibited the ability of p53 to transactivate a p53 responsive promoter element in cotransfection assays. Results The human prostate carcinoma cell line, LNCaP, which possesses a wild-type p53 gene (Carroll et al., 1993), was selected to investigate the inhibition of p53-dependent programmed cell death by bcl-2. Stable bcl-2 expressing LNCaP prostate carcinoma cell lines were generated and con®rmed by Western blotting. Expression of bcl-2 conferred signi®cant resistance to apoptosis induction following g-irradiation compared to control clones as assessed by morphologic and ¯ow cytometric analysis (Figure 1a and b). No signi®cant cell death induction was observed in bcl-2 expressing LNCaP cells up to 48 h following irradiation. Additionally, there was no dierence in cell cycle distribution between LNCaP and LNCaP-bcl-2 cells prior to irradiation and no evidence that the distribution of cells within the cell cycle was altered following irradiation. Approximately 60% of unirradiated control cells reside in G0/G1, 18% in S phase, and 22% in G2/M compared to 59% of unirradiated LNCaP-bcl-2 cells in G0/G1, 21% in S phase, and 20% in G2/M. These values were not signi®cantly dierent from those observed at 30 min, 1 h, 2 h, 4 h and 8 h following irradiation. Apoptosis induced in response to genotoxic damage is considered to be p53-dependent. In LNCaP cells, total cellular p53 protein was induced to approximately equivalent levels in bcl-2 expressing clones and control transfectants within 4 h following 20 Gy g-irradiation (Figure 2a). Western blot analysis using isolated nuclei revealed that levels of p53 protein in the nucleus increased within 2 h of irradiation in control LNCaP,
Bcl-2 inhibits p53 nuclear import A Beham et al
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a
b
Figure 1 (a) Fluorescence microscopic evaluation of cell death induction following irradiation of LNCaP control and bcl-2 transfected cells (LNCaP-bcl-2). Control untreated (upper left), LNCaP-bcl-2 cells untreated (lower left), control cells 24 h following 20 Gy g-irradiation (upper right), and LNCaP-bcl-2 cells 24 h following irradiation (lower right). Cells exhibited the characteristic features of apoptosis are commonly observed in control, but not LNCaP-bcl-2 cells following irradiation. (b) Flow cytometric analysis of cell death induction following 20 Gy of g-irradiation in LNCaP cells. Apoptotic cells (A0) comprise approximately 30% of the LNCaP vector control cell population 8 h following irradiation and 55% in the irradiated LNCaP-bcl-2 cells
but not in bcl-2 expressing LNCaP, cells (Figure 2a). This observation suggests that the nuclear import of p53 following DNA damage may be impaired in the context of high levels of bcl-2 protein. Scanning confocal laser microscopy was used to further characterize p53 nuclear import following irradiation. The inhibition of nuclear p53 import in the bcl-2 cells was con®rmed by confocal microscopy using antibodies which recognize the p53 protein (Figure 2b). Thus, by two independent techniques p53 nuclear import was demonstrated to be significantly inhibited in bcl-2 expressing cells following cell death induction by ionizing radiation. In order to determine whether the transactivating ability of p53 was aected in the context of bcl-2 protein, NIH3T3 cells were transiently transfected with an mdm-2 promoter-luciferase reporter construct which possesses functional p53 binding sites. Luciferase activity increased approximately eightfold following co-transfection with a wild-type, but not mutant, p53 expression plasmid (Figure 2c). Co-transfection of bcl-2 and wild-type p53 expression plasmids resulted in a 2 ± 4-fold decrease in luciferase activity compared to wildtype p53 alone (P40.02). Additional studies were undertaken using the RKO colon carcinoma cell line to assess whether the ability of bcl-2 to inhibit the nuclear import of wild-type p53 was speci®c for the LNCaP prostate cancer cell line. RKO cells possess a wild-type p53 gene (Nagasawa et al., 1995) and also express bcl-2 protein. To downregulate bcl-2 expression in RKO cells, bcl-2 speci®c antisense oligonucleotides were delivered by liposomes. Bcl-2 protein levels were reduced threefold compared to RKO cells treated with empty liposomes or liposomes containing control oligonucleotides (Figure 3a). Confocal microscopy was used to image p53 protein 4 h following 10 Gy of g-radiation in RKO cells treated with liposomes containing antisense bcl-2
oligonucleotides, or control oligonucleotides. RKO cells treated with control oligonucleotides showed that most of the p53 protein remained localized in the cytosol (Figure 3b). In contrast, RKO cells in which bcl-2 had been downregulated by antisense oligonucleotides exhibited high levels of p53 protein within the nucleus and signi®cant (P40.005) cell death induction compared to RKO cell treated with control oligonucleotides (Figure 3c). These ®ndings suggest that the ability of bcl-2 to modulate the import of wild-type p53 protein in response to DNA damage is not cell-type speci®c. Discussion Our results provide new insights regarding the possible basis of bcl-2 inhibition of p53-dependent apoptosis following genotoxic damage. The role of p53 protein in mediating cell cycle arrest and apoptosis has been well documented (Clarke et al., 1993; Kastan et al., 1992; Lowe et al., 1993a,b; Yonish-Rouach et al., 1991). It has also been shown in primary cells and other transformed cell lines that bcl-2 can inhibit p53associated apoptosis (Chiou et al., 1994; Marin et al., 1994). Furthermore, it has been demonstrated that nuclear accumulation of p53 protein is important for mediating p53 cellular responses (Fritsche et al., 1993; Shaulsky et al., 1990). Recent evidence suggests that apoptosis mediated by p53 is dependent on its ability to function as a transcriptional regulator either by transcriptionally activating a death eector program (Miyashita and Reed, 1995), or transcriptional repression of a cellular survival program (Caelles et al., 1994). A role for transactivation-independent, in addition to transactivation-dependent p53 mediated apoptosis has also been described (Caelles et al., 1994; Haupt et al., 1995).
Bcl-2 inhibits p53 nuclear import A Beham et al
Our ®ndings indicate that bcl-2 may inhibit cell death mediated by p53 by impairing p53 nuclear import following genotoxic damage and suggests that the selective modulation of transmembrane tracking may represent a common basis of bcl-2 function. The
basis of these observations does not appear to involve a direct physical interaction between bcl-2 and p53 proteins in that this has not been previously reported. Additionally, we have been unable to co-immunoprecipitate bcl-2 and p53 using available anti-p53 or anti-
c
a Whole Cells CONTROL 0
2
4
Crude Nuclei
BCL-2 0
2
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2
4
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2
4
hrs. γ-rad. p53
β-actin
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Figure 2 (a) Western blot analysis of p53 protein induction and nuclear import following g-irradiation in LNCaP control and LNCaP-bcl-2 cells. Subcon¯uent cultures of control LNCaP and LNCaP-bcl-2 cells were irradiated with 20 Gy. Extracts of whole cells or nuclei isolated from whole cells were prepared 2 and 4 h after irradiation. Equivalent amounts of lysates were analysed by immunoblotting with p53 antibody (Santa Cruz). Corresponding densitometric scans indicates that the amount of p53 protein induced following irradiation is approximately equivalent in whole cell extracts from LNCaP control and LNCaP-bcl-2 cells. However, nuclear accumulation of p53 protein is only observed in nuclei isolated from irradiated LNCaP control cells. (b) Confocal microscopic analysis of p53 subcellular localization following irradiation. LNCaP control (left) and LNCaP-bcl-2 (right) cells were irradiated with 20 Gy, ®xed after 4 h and p53 protein imaged by scanning confocal laser microscopy. Nuclear localization of p53 protein is only observed in LNCaP control cells. (c) Bcl-2 inhibition of transcriptional activation by wt-p53. NIH3T3 cells were transfected with the eector wild-type (P53 WT) or mutant p53 (P53 MUT) plasmid (10 mg), reporter plasmid P2mdm2-Luc (4 mg) and b-galactosidase (b-gal) expression plasmid (3 mg) with or without the bcl-2 expression vector (BCL-2) (20 mg) using the calciumphosphate method. Co-transfection with empty eector vector (VECTOR) served as a negative control. Data represent the fold increase in luciferase activity. Bcl-2 signi®cantly inhibited the ability of wild-type p53 protein to transactivate the mdm2 promoter (*P40.02)
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b Liposomes
control
antisense
a
bcl-2
β-actin
Figure 3 (a) Selective downregulation of bcl-2 protein in RKO colon cancer cells. Western blotting of whole cell extracts (40 mg) were analysed by immunoblotting for bcl-2 protein. A graphic representation of the relative amount of bcl-2 protein after normalization for protein loading is shown. Treatment with antisense bcl-2 oligonucleotides, but not control oligonucleotides or empty liposomes, resulted in a reduction in the amount of bcl-2 protein. (b) Confocal microscopy of p53 protein in irradiated RKO cells treated with control oligonucleotides (top) or antisense-bcl-2 oligonucleotides (bottom). Signi®cant nuclear localization of p53 protein following irradiation is observed only in antisense-bcl-2 treated RKO cells
bcl-2 antibodies (not shown). These results are consistent with observations indicating that bcl-2 may selectively alter import of NF-AT (nuclear factor of activated T cells) following T cell activation (Linette et al., 1995; Shibasaki et al., 1997) and, together with cmyc, in¯uence the subcellular distribution of p53 during cell cycle progression in erythroleukemia cells (Ryan et al., 1994). The results of de Jong et al. (1994), suggest that the ability of bcl-2 to modulate nuclear import of high molecular weight molecules may be mediated by association with components of the nuclear pore complex. Similarly, recent evidence suggests that alterations in the mitochondrial permeability transition pore may represent an early event involved in the initiation of the cell death program and that bcl-2 may inhibit cell death by preventing the pore transition (Zamzami et al., 1996). However, it has also been shown that nuclear events function in some contexts as determinants of apoptotic initiation (Zamzami et al., 1996). Our ®ndings do not enable an assessment of the impact of enforced bcl-2 expression on the ability of wild-type p53 to mediate cell cycle arrest following DNA damage in that growth arrest was not observed in either irradiated control or LNCaP-bcl-2 cells. It has
recently been demonstrated that the cell cycle arrest and apoptotic functions of wild-type p53 are distinct and separable and that the ability of p53 to mediate apoptosis is not dependent on preceding cell cycle arrest (Yonish-Rouach et al., 1993; Abrahamson et al., 1995; Lin and Benchimol, 1995; Little et al., 1995; Chen et al., 1996; Wang et al., 1996; Rowan et al., 1996). Furthermore, it has been shown that overexpression of wild-type p53 does not in all circumstances result in upregulation of p21waf1/cip1 (Jordan et al., 1997) and that even though expression of p21waf1/cip1 may be induced this does not invariably result in cell cycle arrest (Sheikh et al., 1996; Little et al., 1995). These seemingly disparate ®ndings have prompted the speculation that the contribution of p53 to cell cycle arrest and apoptosis is ultimately determined by a complex network of signaling events that may be cell type or context dependent (Yonish-Rouach, 1996). Together, these results suggest that a common feature of bcl-2 function, irrespective of its subcellular localization, may be the ability to selectively modulate transmembrane tracking of signal molecules necessary for the mediation of cell death. Therefore, we speculate that this `gatekeeper' function may be an essential component for cell death suppression by bcl-2.
Bcl-2 inhibits p53 nuclear import A Beham et al
Whether the mechanism of the `gatekeeper' involves a physical association of bcl-2 with existing pore complexes within these membrane compartments (de Jong et al., 1994), or is mediated indirectly by competitive interactions with proteins which may be necessary for transmembrane tracking (Elkind et al., 1995; Naumovski and Cleary, 1996; Shibasaki et al., 1997) or modi®cation of the intracellular ion environment (Greber and Gerace 1995; Marin et al., 1996) remains to be completely elucidated. It is, therefore, conceivable that bcl-2 may function to selectively modulate import events directly at the level of the nuclear pore complex itself or function to modify requisite activation events upstream of membrane translocation. Alternatively, recent models proposed for the three-dimensional structure of the bcl-2 family member, bcl-xL, indicates that these proteins resemble certain bacterial pore forming proteins (Muchmore et al., 1996) and suggest that bcl-2 family members may, thereby, function as their own eector proteins.
Materials and methods Irradiation and calcein-AM staining Cells were preloaded with calcein-AM (Molecular Probes, Eugene, OR) at 1 mg/ml for 20 min in RPMI containing 10% FBS. Cells were given 20 Gy of radiation using a 137Cs g-source and calcein-generated ¯uorescence was visualized using epi¯uorescence optics and a FITC ®lter (530 nm emission). The 20 Gy dose of radiation was selected as the minimum dose required to induce maximum levels of apoptosis in LNCaP cells. Flow cytometric analysis Flow cytometric analysis of cell cycle and cell death induction in LNCaP cells. Single cell suspensions were ®xed in 70% ethanol and incubated with 50 mg/ml propidium iodide (PI) and 20 mg/ml RNAse for 15 min at 378C. Flow analysis was done with a EPICS Pro®le I at 488 nm excitation and collected for PI ¯uorescence using Elite Software 4.0 (Coulter Corp, Miami, FL) and the Multi Cycle DNA Analysis program software (Phoenix Flow Systems, San Diego, CA). Transfections and Luciferase assays LNCaP cells were transfected with the splenic focus forming virus expression plasmid with, or without (control), the bcl-2 cDNA insert as previously described (Marin et al., 1996; Tu et al., 1995). The eector plasmids LTRXA and LTRKH expression plasmids have been described previously (Dee et al., 1993) and represent wild-type and mutant p53, respectively. The reporter plasmid P2mdm2-Luc was made by cloning a 1 kb XhoIfragment containing the p53 responsive element from the mouse mdm2 gene into the SmaI site of the pA3-luciferase plasmid. NIH3T3 cells were plated at a density of 0.5610 6 cells per plate 24 h before transfection. The eector wildtype or mutant p53 plasmid (10 mg), reporter plasmid P2mdm2-Luc (4 mg) and b-galactosidase (b-gal) expression plasmid (3 mg) were co-transfected with or without bcl-2 vector (20 mg) following the calcium-phosphate method. The total amount of DNA transfected was normalized adding p-GEM plasmid up to 37 mg of total DNA for all the transfections. At 48 h after the transfection the cells were harvested. Extracts were made and assayed for luciferase activity.
Immuno¯uorescence staining and confocal microscopy LNCaP control and LNCaP-bcl-2 cells were grown on laminin coated cover slides and irradiated with 20 Gy. After 4 h, cells were washed twice with PBS, then ®xed in 4% paraformaldehyde for 10 min and washed twice in PBS. Cells were blocked with 10% goat serum in PBS, incubated with p53 (AB-2, Calbiochem) antibody in 10% goat serum (1 : 75), washed twice, and incubated with ¯uorescein isothiocyanate (FITC)-labeled secondary antibody in 10% goat serum (1 : 200). Imaging was done using a Zeiss scanning confocal laser microscope. RKO cells were grown on laminin coated cover slides and incubated with liposomal oligonucleotide formulations (described below) at ®nal concentration of 10 m M at 378C in a 5% CO2 incubator for 3 days. Four hours after irradiation with 10 Gy cells were washed twice with PBS, then ®xed in 4% paraformaldehyde for 10 min and washed twice in PBS. Cells were blocked with 10% goat serum in PBS, incubated with p53 (AB-2, Calbiochem) antibody in 10% goat serum (1 : 75), washed twice and incubated with ¯uorescein isothiocyanate (FITC)-labeled secondary antibody in 10% goat serum (1 : 200). Confocal microscopy was done using a Zeiss scanning laser confocal microscope. Antisense and DNA methods P-ethoxy-oligonucleotides, a non-ionic and nuclease-resistant phosphodiester analog, were purchased from Oligo Therapeutics (Willsonville, OR). An oligonucleotide speci®c for the translation initiation site of human bcl-2 mRNA: 5'CAGCGTGCGCCATCCTTC-3' was used as antisense oligonucleotide. The control oligonucleotide used was a scrambled version of Bcl-2 antisense oligonucleotide 5'ACGGTCCGCCACTCCTTCCC-3'. P-ethoxy-oligonucleotides, dissolved in DMSO, were added to phospholipids (Avanti Polar Lipids, Alabaster, AL) in the presence of excess tert-butanol. The mixture was frozen in a dry ice/ acetone bath, lyophilized overnight and hydrated with 0.9% saline at a ®nal oligonucleotide concentration of 0.1 mmol/l. Empty liposomes were prepared identically as above, except that oligonucleotides were not included in the preparation. 0.256105 cells/ml were seeded in a 24-well plate in 0.5 ml of the respective medium. Cells were incubated with antisense, control oligonucleotides and empty liposomes at ®nal concentration of 10 mM at 378C in a 5% CO2 incubator for 3 days. Western blotting of whole cell extracts (40 mg) of control and bcl-2 transfected clones were analysed by immunoblotting for bcl-2 protein using an anti-bcl-2 monoclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, California). Analysis of protein expression in cell and nuclear extracts Western blot analysis of p53 protein induction and nuclear import following g-irradiation. Subcon¯uent cultures of control LNCaP and LNCaP-bcl-2 cells were irradiated with 20 Gy. Extracts of nuclei were prepared by scraping cell monolayers into hypotonic lysis buer (100 mM HEPES, pH 7.4. 1.5 mM MgCl2, 10 mM KCl, 0.5 mM b-mercaptoethanol and 5 ng/ml leupeptin). After 10 min on ice, NP40 was added to 0.625% and the crude nuclear pellet was recovered by centrifugation at 2000 g for 5 min. The nuclear pellets were lysed in SDS ± PAGE sample loading buer. Extracts of nuclei and whole cells were prepeared 2 and 4 h after irradiation. Equivalent amounts of lysates were analysed by immunoblotting with p53 antibody (Santa Cruz). Extracts of nuclei were prepared by scraping cell monolayers into hypotonic lysis buer (100 mM HEPES, pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM b-mercaptoethanol and 5 ng/ml leupeptin). After 10 min on ice, NP-40 was added to 0.625% and the crude nuclear pellet was recovered by centrifugation at 2000 g for 5 min. The nuclear pellets were lysed in SDS ± PAGE sample loading buer.
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Acknowledgements Supported by ACS grant DHP-156 and CaP CURE (The Association for the Cure of Cancer of the Prostate) and the Pew Scholars Program in the Biomedical Sciences. MCM
was supported by NIH Predoctoral Fellowship CA09255. AB was supported by the Deutsche Forschungsgemeinschaft.
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