Caspase 3 is an essential death factor for the Fas-mediated cell death, and its inactivation in cells is initiated by an interaction with p21 on mitochondria or with IAP family member ILP. Survivin is also a member of IAP family and is specifically expressed during embryogenesis and in tumor cells and suppresses cell death signaling. In our current study, we demonstrated that Survivin translocation into the nucleus is dependent on Fas stimulation and cell proliferation. Survivin also interacts with the cell cycle regulator Cdk4, leading to Cdk2/Cyclin E activation and Rb phosphorylation. As a result of Survivin/Cdk4 complex formation, p21 is released from its complex with Cdk4 and interacts with mitochondrial procaspase 3 to suppress Fas-mediated cell death. Here, we propose that Survivin supports procaspase 3/p21 complex formation as a result of interaction with Cdk4 resulting in suppression of cell death signaling.
Cell death is an essential phenomenon for cell homeostasis, as well as cell growth, and has been well documented during embryonic and postembryonic development (Wyllie et al., 1980; Nagata 1997). There are two distinct processes leading to cell death known as apoptotic cell death and necrotic cell death (Wyllie et al., 1980). Apoptotic cell death is accompanied by the condensation and/or fragmentation of nuclei, apoptotic body formation and chromosomal DNA fragmentation into 180 bp oligomers (Wyllie et al., 1980). Multiple studies have demonstrated the important role of apoptotic cell death in various disease states and in physiological cell death (Nagata and Golstein, 1995) and many factors involved with the death signaling has been identified.
Fas is a transmembrane protein belonging to the tumor necrosis factor/nerve growth factor receptor family (Nagata and Golstein, 1995) and transduces the death signaling upon stimulation by Fas ligand or an agonistic Fas antibody, such as the CH-11 clone (Yonehara et al., 1989). The molecular mechanism of Fas-mediated apoptosis has been extensively investigated. Caspase is the nomenclature that refers to the interleukin-1β converting enzyme (ICE)/CED-3 cysteine proteinase family (Alnemri et al., 1996). During death induction, sequential activation of the ICE and CPP32 subfamilies has been reported (Enari et al., 1996; Shimizu et al., 1996; Suzuki, 1997; Suzuki et al., 1997a,b, 1998a) and this phenomenon is known as the ‘ICE cascade’. At present, ten genes has been identified as caspase family, and the CPP32 subfamily, including caspase 3 (CPP32/Yama/Apopain and ref. Fernandes-Alnemri et al., 1994; Nicholson et al., 1995) and caspase 8 (FLICE/MACH and ref. Boldin et al., 1996; Muzio et al., 1996), in partcular acts as the dominant regulator in the death signaling. Therefore, the regulation of CPP32 subfamily activation is an especially important focus for cell death research.
Among CPP32 subfamily, caspase 3 is especially important for the understanding of apoptotic cell death because of its variant substrate-specificity. Cytoplasmic serine proteinase (Suzuki et al., 1997b), caspase 8 (Thornbery et al., 1997) and/or cytotoxic T lymphocyte-derived granzyme B (Darmon et al., 1995) proteolyses caspase 3 for its activation, and activated caspase 3 proteolyses and/or activates poly (ADP-ribose) polymerase (Tewari et al., 1995), lamin (Lazebnik et al., 1995) and/or DFF (Liu et al., 1997) to induce apoptotic cell death. Recently, we reported that the cell cycle regulator p21 and IAP gene family ILP act as inactivators of caspase 3 (Suzuki et al., 1998b, 1999a). p21 is especially unique in that it interacts with only procaspase 3 by each N-terminal sequence on mitochondria and suppresses its activation by the masking of cytoplasmic serine proteinase-cleaving site (Suzuki et al., 1998b, 1999a,b). Thus, activation of caspase 3 is regulated by p21, and the procaspase 3/p21 complex formation is an essential system for the cell death such that cell survival is a result of cell death suppression (Suzuki et al., 1998b).
Survivin is also a member of the IAP family, and its specific expression is encountered during embryogenesis and in tumor cells (Ambrosini et al., 1997; Adida et al., 1998). Recently, a direct interaction between Survivin and caspase family members, especially caspase 3 and 7, was reported with a cell free system, however, the molecular mechanism resulting in death suppression by Survivin is not clarified yet. In the present study, therefore, we investigated the molecular machinery of Survivin-initiated cell death suppression.
Fas Ab treatment initiates the nuclear translocation of Survivin
HepG2 cells shows the Fas-mediated cell death only in the presence of actinomycin D. Interestingly, we demonstrated that Fas Ab treatment without actinomycin D induced increases in the MTT value and cell number (Figure 1a). Following Fas Ab-induced slight cell proliferation, Survivin translocated into nuclei (Figure 1b), suggesting a possible interaction of Survivin with cell cycle-associated factors. In contrast, Fas Ab-treatment did not induce any changes in expressions of actin (cytoplasmic protein marker) and lamin (nuclear protein marker). Co-immunoprecipitation analysis revealed that Survivin interacted with Cdk4 among tested cell cycle-associated factors (Figure 1c).
Survivin interacts with Cdk4, rather than caspase 3
An interaction of Survivin with caspase 3 has been reported (Tamm et al., 1998). In our current study, cell free experiment using each recombinant protein revealed that caspase 3 interacted with both Survivin and ILP, same as previous reports (Suzuki et al., 1998b; Tamm et al., 1998) (Figure 2a). However, co-immunoprecipitation analysis using cellular proteins from Fas Ab-treated HepG2 demonstrated that Survivin interacted with Cdk4, rather than caspase 3 (Figure 2b). In addition, an expression of Survivin was disappeared before caspase 3 activation during Fas-mediated cell death (Figure 2c). Interestingly, Survivin delayed the Fas-mediated cell death in HepG2 cells with the delay of p21 disappearance during Fas-mediated cell death (Figure 2d).
Survivin initiates procaspase 3/p21 complex formation as a result of an interaction with Cdk4
We examined an effect of nuclear translocation of Survivin induced by Fas Ab treatment in the formation of procaspase 3/p21 complex. When cells were treated with Fas Ab, the procaspase 3/p21 complex formation on mitochondria was accelerated (Figure 3). In addition, Cdk4 which expression was not affected by Fas Ab treatment, interacted dominantly with p21 before Fas Ab treatment, however, Cdk4 bound Survivin upon the stimulation of Fas Ab, rather than p21 (Figure 4). The nuclear translocation of Survivin stimulated by Fas Ab treatment accelerated procaspase 3/p21 complex resulting from mitochrondrial translocation of p21 initiated by Survivin/Cdk4 complex formation. However, microinjection of Survivin Ab into cells suppressed these phenomenon (Figure 5a).
HepG2 cells showed the Fas-mediated cell death only in the presence of actinomycin D. When HepG2 cells were microinjected with GST-Survivin, Fas-mediated cell death was partially induced even in the absence of actinomycin D (Figure 5b).
Influence of Survivin-nuclear translocation in cell cycle
Since Fas Ab treatment induces nuclear translocation of Survivin, we investigated cell cycle-associated factor in Fas Ab-treated HepG2 cells. When HepG2 cells were treated with Fas Ab in the absence of actinomycin D, p27 interacting with Cdk2/Cyclin E complex reduced (Figure 6a), and that interacting with Cdk4/Cyclin D1 complex increased (Figure 6b). Following these events, we demonstrated up-regulation of phosphorylated Rb (Figure 6c).
When human hepatoma HepG2 cells were reacted with agonistic Fas antibody (Fas Ab: CH-11 clone and ref. Yonehara et al., 1989), Fas-mediated cell death was observed only in the presence of the de novo protein synthesis inhibitor actinomycin D, consistent with our previous reports (Suzuki et al., 1998b, 1999a,b). Interestingly, Fas Ab-treatment showed the trend of cell proliferation. We additionally demonstrated that Survivin translocated into nuclei and interacted with only cyclin dependent kinase 4 (Cdk4). These observations, namely the nuclear translocation of Survivin and its interaction with Cdk4, are first findings.
An interaction of Survivin with the death mediator caspase 3 in a cell free system has also been reported (Tamm et al., 1998). In the present study, human recombinant active caspase 3 interacted with GST-Survivin and GST-ILP in a cell free system. However, co-immunoprecipitation analysis with HepG2 cellular proteins revealed that Survivin interacted with Cdk4, rather than activated caspase 3. In addition, cellular expression of Survivin disappeared before caspase 3 activation as monitored by poly (ADP-ribose) polymerase (PARP) cleavage (Nicholson et al., 1995), during Fas-mediated cell death. While the caspase 3 direct suppressors, p21 or ILP, completely prevent Fas-mediated cell death, Survivin delays it. This is consistent with the partial prevention of Fas-mediated cell death by over-expressed Survivin (Tamm et al., 1998) and also suggests that Survivin does not directly suppress caspase 3. Interestingly, Survivin suppressed the disappearance of p21 by actinomycin D treatment. We suggest that Survivin prevents the loss of p21 and this delay initiates the partial prevention of Fas-mediated cell death. Survivin may be indirectly involved with the caspase 3 inactivation system through its interaction with Cdk4 and possible influence on the procaspase 3/p21 complex.
Recently, we reported that the cell cycle regulator p21 interacts with mitochondrial procaspase 3 through NH2-terminal amino acid sequences to suppress Fas-mediated cell death (Suzuki et al., 1998b, 1999a,b). As a cell cycle regulator, p21 interacts with Cdk4 (Luo et al., 1995). We investigated whether the Survivin/Cdk4 complex influence procaspase 3/p21 complex formation and demonstrated that Fas Ab treatment induced translocation of Survivin to nuclei and procaspase 3/p21 complex formation in mitochondria. As indicated above, p21 interacted with Cdk4 to induce cell cycle arrest, and we showed Survivin also interacted with Cdk4 to initiate cell cycle entry. In addition, Cdk4 interacted predominantly with Survivin after Fas Ab treatment. These observations suggest that nuclear-translocated Survivin interacts with Cdk4 to release p21 from Cdk4/p21 complex.
The overall data provide evidence regarding the mechanism of death suppression by Survivin. We found that Survivin translocates into nuclei upon stimulation of Fas and cell proliferation, as well as Survivin interactions with Cdk4 that release p21 from the Cdk4/p21 complex. Our present results strongly suggest that Survivin is necessary for the procaspase 3/p21 complex formation to resist Fas-mediated cell death. Thus, it became of interest to examine effects of a functional loss of Survivin. When antibody to Survivin was microinjected into HepG2 cells for the neutralization of endogenous Survivin, a decrease was observed in mitochondrial p21 as well as reduced formations of procaspase 3/p21 complex and Survivin/Cdk4 complex. As indicated above, HepG2 cells shows the Fas-mediated cell death only in the presence of actinomycin D, and we reported that this resistance is due to caspase 3 inactivation by p21 and/or ILP and that the treatment with actinomycin D induced decreases of p21 and ILP (Suzuki et al., 1998b, 1999a,b). In the current study, we demonstrated that Survivin influenced procaspase 3/p21 complex formation and the neutralization of Survivin induced partially Fas-mediated cell death in HepG2 cells even in the absence of actinomycin D, suggesting that the partial induction of Fas-mediated cell death is initiated by down-regulation of procaspase 3/p21 complex formation by Survivin neutralization. We also suggest that the partial induction, not complete, of Fas-mediated cell death is due to the suppression of activated caspase 3 by ILP. These results indicate that Survivin initiates procaspase 3/p21 complex formation as a result of interaction with Cdk4.
Survivin is known as a tumor-specific factor (Ambrosini et al., 1997; Adida et al., 1998; Tamm et al., 1998). Survival of the tumor cell results from cell proliferation (cell cycle entry) and resistance to cell death signaling. In the current study, we demonstrated that the phosphorylation of Rb which is S phase entry-inducer (Buchkovich et al., 1989; Chen et al., 1989; DeCaprio et al., 1989; Lin et al., 1991; Zhu et al., 1996), was induced according to nuclear translocation of Survivin upon stimulation of Fas. Nuclear translocated Survivin interacting Cdk4 accelerated p27 estrangement from Cdk2/Cyclin E complex which induces Rb phosphorylation. During this Rb phosphorylation pathway, p21 was released from Cdk4/Cyclin D1 complex. Recently, p21 and p27 act for the stabilization of Cdk/Cyclin complex (Cheng et al., 1999; Sherr and Roberts, 1999). In the current study, we could not clarify the role of p21 and p27 for Cdk/Cyclin complex, however, our results indicate that p21 is also necessary for the resistance to Fas-mediated cell death and is supplied from Rb phosphorylation pathway accelerated by Survivin. On the basis of our present data, our current working model for Survivin and cell death resistance mechanisms is illustrated schematically in Figure 6d. Survivin translocates into the nucleus and interacts with Cdk4. As a result of Survivin/Cdk4 complex formation, cell cycle entry is initiated and p21 is released during Rb phosphorylation pathway. The released p21 translocates to mitochondria and forms a complex with procaspase 3 with NH2-terminal amino acid sequence. The resultant procaspase 3/p21 complex formation acts as to resist Fas-mediated cell death. In this way, tumor cells acquire the necessary cell survival mechanisms of cell cycle entry and resistance to cell death signaling.
Materials and methods
Cell line and culture
Human hepatoma HepG2 cells were supplied by Dr Y Tsujimoto (Hasegawa et al., 1996) and were maintained in RPMI1640 medium (Gibco BRL., MD, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco BRL, MD, USA) in a humidified atmosphere of 5% CO2 and 95% air.
Preparation of Survivin antibody
A polyclonal antibody to the anti-Survivin was generated in rabbits using a synthetic peptide (A3PTLPPAWQPFLKDHRI19C) and purified by affinity chromatography on a peptide-Sepharose matrix (5 mg/ml peptide) with elution of specific IgG in 1 mol/l glycine, pH 2.5.
Preparation of GST-Survivin
The cDNA was amplified using RT–PCR from total RNA of SW480 colorectal cancer cell line. The 5′-sense primer and the 3′-antisense primer were 5′-CCGGGATCCATGGGTGCCCCGACGTTG-3′ and 5′-CGCGAATTCAGAGGCCTCAATCCATGG-3′, respectively. The amplified fragments were first subcloned into the pCR2.1 vector. The cloned cDNA was excised by digestion with BamHI and EcoRI and ligated into the pGEX 4T-1 vector. The three constructs were used to transform competent Escherichia coli BL21(DE3) cells and cultured on Luria-Bertani (LB) plates at 37°C for 12 h. Single colony cultures were grown at 37°C until the OD600 nm reached 0.6–0.8, induced with isopropyl1-thio-5′-D-galactoside (0.1 mM) and the cultures grown for an additional 4 h. The cell pellets were homogenized and sonicated. After centrifugation, the supernatants were clarified by filtration through a 0.45 m filter, and soluble GST fusion proteins were purified from the supernatant by affinity chromatography on glutathion-Sepharose.
Preparation of ILP antibody
pGEX5X-3 was digested by BamHI, blunted with Klenow fragment and then self-ligated. The resulting plasmid was named pGEX5X-3′. The myc-epitope tagged ILP cDNA (pcDNAs-mycILP) was kindly donated by Dr CB Thompson. A mycILP fragment was isolated by EcoRI digestion and subcloned into pGEX5X-3′. GST-mycILP was expressed in E. coli and purified on Glutathione Sepharose 4B columns. An antigen emulsion was made from protein (100 μg) and complete Freund adjuvant (for primary immunization) or incomplete Freund adjuvant (for booster immunization) by using ultrasonicator. Female 8-week-old Balb/c mice were immunized subcutaneously. Booster immunizations were given 2 and 5 weeks later, and then blood was collected 1 week after the final booster immunization.
Preparation of fractionated proteins
Fractionated proteins were collected by a previously described method (Suzuki et al., 1999b). Cells were collected and washed with ice-cold PBS, and then suspended in buffer I (2 mM EDTA and 10 mM Tris-HCl, pH 7.5). After incubation on ice for 10 min, an equal volume of buffer II (0.5 M sucrose, 0.1 M KCl, 10 mM MgCl2, 2 mM CaCl2, 2 mM EDTA, 10 mM Tris-HCl, pH 7.5) was added. The nuclear-rich fraction was pelleted by centrifugation (2700 r.p.m./10 min). The supernatant was removed to a separate tube and again centrifuged (43 000 r.p.m./90 min). The supernatant was collected as the cytosol-rich fraction, and the pellet was dissolved in buffer III (8 mM CHAPS, 150 mM NaCl, 0.1 M sucrose, 2 mM EDTA, 10 mM Tris-HCl, pH 7.5) and incubated at 4°C for 2 h. The membrane-rich fraction was then collected by centrifugation (43 000 r.p.m./60 min). Mitochondria were collected using a previously described method (Fleisher and Kervina 1974), and its proteins were extracted with 1% NP40-containing PBS. Each fractionation was separated by 5–20% SDS–PAGE and p21-expression was detected by immunoblotting analysis with anti-human p21 antibody and ECL detection system (Amersham, UK).
Antibody for Survivin was diluted in PBS to 1 mg/ml and microinjection of HepG2 cells was performed with a Eppendorf Transjector 5246. Microinjection was performed at 100 hPa for 3 s. In the present study, no abnormalities were observed during the first week after the cells were microinjected with PBS.
Assay of cell viability
Cell viability of each group was assessed by the Hoechst 33342/propidium iodide (PI) staining procedure as previously described (Hasegawa et al., 1996) with some modifications (Suzuki et al., 1997a,b). Hoechst 33342 and PI were purchased from Molecular Probes, Inc. (OR, USA). After treatment, cells were collected and stained with Hoechst 33342 and then observed by fluorescence microscopy. Cell viability was indicated by the ratio of cells carrying intact nuclei to total cells (about 5000 cells).
Cell viability was measured with the MTT assay as described previously (Mossman, 1983). After treatment, 10 μl of PBS-diluted MTT (5 mg/ml) was added to each well, and then the plate was incubated for 4 h, followed by the addition of DMSO. The plate was maintained at room temperature for 5 min, and the absorbance ratio A570/A690 was read on a microtiter plate reader (Titerteck, Fukuoka, Japan).
Proteins were prepared for immunoblotting and immunoprecipitation analysis by lysis of cells with 1% NP-40 containing buffer for 30 min. All procedures were carried out at 4°C. Proteins were collected by the centrifugation at 15 000 r.p.m. for 15 min. Protein concentrations were determined by a method described previously (Smith et al., 1985; Hill and Sraka 1988) using DC protein assay kit (Bio-Rad) with bovine serum albumin as standard.
Sample proteins separated by SDS–PAGE were transferred onto nitrocellulose membranes with a semi-dry blotting system. The membranes were blocked with PBS containing 5% (w/v) skim milk at room temperature for 1 h, washed with a mixture of PBS and 0.05% Tween 20 (Sigma, Tween-PBS), and then incubated overnight at room temperature with each antibody diluted with PBS. After washing with Tween-PBS, the membranes were incubated with a 1000-fold diluted biotinylated anti-mouse IgG or anti-rabbit IgG antibody (Bio Source, CA, USA), washed with Tween-PBS, and then incubated with avidin-HRP (Vector Lab., CA, USA) at room temperature for 1 h. The membranes were washed with Tween-PBS and then developed with the ECL system.
Antibody was mixed with 1 mg protein from each cell extract for 30 min. Protein A-sepharose was then added and incubated for 2 h. The immunoprecipitates were heated in SDS sample buffer and separated on 5–20% polyacrylamide gels. After transfer, the membranes were immunoblotted, washed with Tween-PBS and developed using the ECL system (Amersham, Buckinghamshire, UK).
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We thank Dr Y Tsujimoto for human hepatoma HepG2 cells, Dr CB Thompson for the ILP cDNA, Dr HR Horvitz for the pET21b-hcpp32-His6 clone plasmid, and Dr H Matsushime for pGST-p21. The preparation of this manuscript was supported by the Idest Inc., Edmond, OK, USA.
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Cite this article
Suzuki, A., Ito, T., Kawano, H. et al. Survivin initiates procaspase 3/p21 complex formation as a result of interaction with Cdk4 to resist Fas-mediated cell death. Oncogene 19, 1346–1353 (2000). https://doi.org/10.1038/sj.onc.1203429
- Procaspase 3/p21 complex
- Fas-mediated cell death
- cell survival
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