Direct Interaction between Survivin and Smac/DIABLO Is Essential for the Anti-apoptotic Activity of Survivin during Taxol-induced Apoptosis
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m300957200
ISSN1083-351X
AutoresZhiyin Song, Xuebiao Yao, Mian Wu,
Tópico(s)CRISPR and Genetic Engineering
ResumoSurvivin is a member of the inhibitor of apoptosis protein (IAP) family that has been implicated in both apoptosis inhibition and cell cycle control. However, its inhibitory mechanism and subcellular localization remain controversial. In this report, we provided evidence for the first time that Survivin physically interacts with Smac/DIABLO both in vitro and in vivo. A point mutation (D71R) in the baculovirus IAP repeat motif and a C-terminal deletion mutant (Surv-BIR) of Survivin fail to bind to Smac/DIABLO and abrogate its ability to inhibit apoptosis. The N-terminal of mature Smac/DIABLO is absolutely required for Survivin·Smac complex formation. Subcellular distributions of Survivin and Smac/DIABLO showed that they co-localized within the cytosol during interphase. In addition, Survivin was found to be incapable of binding to caspase. We also identified that the co-presence of Smac/DIABLO and XIAP was required for Survivin to inhibit caspase cleavage in a cell-free system. In conclusion, our results provide the first evidence that the interaction between Smac/DIABLO and Survivin is an essential step underling the inhibition of apoptosis induced by Taxol. Survivin is a member of the inhibitor of apoptosis protein (IAP) family that has been implicated in both apoptosis inhibition and cell cycle control. However, its inhibitory mechanism and subcellular localization remain controversial. In this report, we provided evidence for the first time that Survivin physically interacts with Smac/DIABLO both in vitro and in vivo. A point mutation (D71R) in the baculovirus IAP repeat motif and a C-terminal deletion mutant (Surv-BIR) of Survivin fail to bind to Smac/DIABLO and abrogate its ability to inhibit apoptosis. The N-terminal of mature Smac/DIABLO is absolutely required for Survivin·Smac complex formation. Subcellular distributions of Survivin and Smac/DIABLO showed that they co-localized within the cytosol during interphase. In addition, Survivin was found to be incapable of binding to caspase. We also identified that the co-presence of Smac/DIABLO and XIAP was required for Survivin to inhibit caspase cleavage in a cell-free system. In conclusion, our results provide the first evidence that the interaction between Smac/DIABLO and Survivin is an essential step underling the inhibition of apoptosis induced by Taxol. Among the regulators of apoptosis, considerable interest has been focusing on the inhibitors of apoptosis protein (IAP) 1The abbreviations used are: IAP, inhibitor of apoptosis protein; BIR, baculovirus IAP repeat; GST, glutathione S-transferase; Ab, antibody; GFP, green fluorescence protein; EGFP, enhanced GFP; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; cyto c, cytochrome c; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand. 1The abbreviations used are: IAP, inhibitor of apoptosis protein; BIR, baculovirus IAP repeat; GST, glutathione S-transferase; Ab, antibody; GFP, green fluorescence protein; EGFP, enhanced GFP; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; cyto c, cytochrome c; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand. family, which were first identified as negative regulators in programmed cell death characterized by the presence of one to three copies of the baculovirus IAP repeat (BIR) domain (1Crook N.E. Clem R.J. Miller L.K. J. Virol. 1993; 67: 2168-2174Crossref PubMed Google Scholar). However, anti-apoptotic function in physiological cell death has not been established for all IAPs, because some members in this family play essential roles in cell division, rather than merely acting as the regulators of apoptosis (2Uren A.G. Wong L. Pakusch M. Fowler K.J. Burrows F.J. Vaux D.L. Choo K.H. Curr. Biol. 2000; 10: 1319-1328Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). Survivin (16.5 KDa) is a special member of the inhibitor-of-apoptosis proteins (IAPs) family containing a single BIR and lacking a RING finger motif. It is mostly expressed in the vast majority of tumors or in the embryonic development, but not in normal adult tissues (3Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (2997) Google Scholar, 4Adida C. Crotty P.L. McGrath J. Berrebi D. Diebold J. Altieri D.C. Am. J. Pathol. 1998; 152: 43-49PubMed Google Scholar). Survivin is cell cycle-regulated and predominantly expressed in the G2/M phase (5Li F. Ambrosini G. Chu E.Y. Plescia J. Tognin S. Marchisio P.C. Altieri D.C. Nature. 1998; 396: 580-584Crossref PubMed Scopus (1719) Google Scholar). The degradation of Survivin is believed to be through the ubiquitin-proteasome pathway (6Zhao J. Tenev T. Martins L.M. Downward J. Lemoine N.R. J. Cell Sci. 2000; 113: 4363-4371Crossref PubMed Google Scholar). Accumulating evidence supports the idea that Survivin is a bifunctional protein that acts as a cell division regulator (7Fraser A.G. James C. Evan G.I. Hengartner M.O. Curr. Biol. 1999; 9: 292-301Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 8Li F. Ackermann E.J. Bennett C.F. Rothermel A.L. Plescia J. Tognin S. Villa A. Marchisio P.C. Altieri D.C. Nat. Cell Biol. 1999; 1: 461-466Crossref PubMed Scopus (553) Google Scholar, 9Miller L.K. Trends. 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Zhou Q. Sun C. Fesik S.W. Liddington R.C. Salvesen G.S. Cell. 2001; 104: 791-800Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). Verdecia et al. (18Verdecia M.A. Huang H. Dutil E. Kaiser D.A. Hunter T. Noel J.P. Nat. Struct. Biol. 2000; 7: 602-608Crossref PubMed Scopus (301) Google Scholar) and Banks et al. (19Banks D.P. Plescia J. Altier D.C. Chen J. Rosenberg S.H. Zhang H. Ng S.C. Blood. 2000; 96: 4002-4003Crossref PubMed Google Scholar) have demonstrated that Survivin is unable to directly bind to caspase-3 in vitro and does not inhibit caspase-3 activity. Wang and colleagues identified a novel mitochondrial-associated protein Smac/DIABLO that was able to interact with IAPs (20Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2891) Google Scholar, 21Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1962) Google Scholar) and may help explain the inhibitory function of Survivin.Smac/DIABLO is a mitochondria protein but is released into the cytosol in response to some of apoptotic stimuli, including UVB-irradiation, etoposide, or glucocorticoids (20Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2891) Google Scholar, 21Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1962) Google Scholar, 22Chauhan D. Hideshima T. Rosen S. Reed J.C. Kharbanda S. Anderson K.C. J. Biol. Chem. 2001; 276: 24453-24456Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 23Ekert P.G. Silke J. Hawkins C.J. Verhagen A.M. Vaux D.L. J. Cell Biol. 2001; 152: 483-490Crossref PubMed Scopus (162) Google Scholar). After mitochondria import, the N terminus of precursor Smac/DIABLO is removed by limited proteolysis to generate a mature form of the molecule (20Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2891) Google Scholar, 27Srinivasula S.M. Datta P. Fan X.J. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Mature Smac/DIABLO was found to promote caspase activation by binding and neutralizing the IAPs, including XIAP, CIAP-1, and CIAP-2 (20Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2891) Google Scholar, 21Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1962) Google Scholar, 23Ekert P.G. Silke J. Hawkins C.J. Verhagen A.M. Vaux D.L. J. Cell Biol. 2001; 152: 483-490Crossref PubMed Scopus (162) Google Scholar). Numbers of reports confirmed that mature Smac/DIABLO interacts with BIR2 and BIR3 of XIAP, and the N terminus of mature Smac/DIABLO is absolutely required for this interaction (24Chai J. Du C. Wu J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (701) Google Scholar, 26Wu G. Chai J. Suber T.L. Wu J.W. Du C. Wang X. Shi Y. Nature. 2000; 408: 1008-1012Crossref PubMed Scopus (702) Google Scholar, 27Srinivasula S.M. Datta P. Fan X.J. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 28Srinivasula S.M. Hegde R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R.A. Robbins P.D. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (852) Google Scholar, 29Chai J. Shiozaki E. Srinivasula S.M. Wu Q. Datta P. Alnemri E.S. Shi Y. Dataa P. Cell. 2001; 104: 769-780Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). However, whether Smac/DIABLO physically interacts with Survivin has yet to be fully characterized.In this report, we investigated the effect of interactions between Survivin and Smac/DIABLO on the apoptosis induced by Taxol (paclitaxel). We have demonstrated that Taxol is able to trigger apoptosis significantly in HeLa cell, resulting in the activation of cellular caspases-3, -7, and -9 and release of Smac/DIABLO and cytochrome c from the mitochondrial. Ectopic expression of Survivin was able to significantly block the Taxol-induced cell death. Our GST pull-down binding assay and immunoprecipitation demonstrate for the first time that Survivin directly interacted with Smac/DIABLO but not with caspases. We also found mutant Survivin with a single amino acid change at residue Asp-71 (D71R) or truncated Survivin lacking the C terminus (residues 1–97) failed to bind to Smac/DIABLO and abrogated its ability to inhibit apoptosis. On the contrary, these two Survivin mutants displayed some apoptotic effects. In addition, we have shown that wild type Survivin does not interact with Smac mutants M-Smac (a methionine was added to the begin of mature Smac) or Δ74Smac (residues 75–239). We examined subcellular distributions of Survivin and Smac/DIABLO within living cells at different stages of the cell cycle and found that Survivin and Smac are co-localized in the cytosol during interphase, and when cells enter into M phase Survivin acts as a chromosome passenger protein. In addition, by using the cell-free system, we were able to demonstrate that both Smac/DIABLO and XIAP are required in order for inhibition of caspase cleavage by Survivin. Combined, our data strongly support our proposed hypothetical mode for Survivin in the inhibition of apoptosis. During apoptosis Survivin binds to Smac, which is released from induced mitochondria. Therefore, by reducing Smac/DIABLO antagonism to IAPs, such as XIAP, the free XIAP directly interacts with caspases and cell death is blocked.MATERIALS AND METHODSOligonucleotides—The sequences of the oligonucleotides used in this study are listed as follows. P1, 5′-CGGGATCCGCATGGGTGCCCCGACGTTG-3′; P2, 5′-CGGTCGACCCATCCATGGCAGCCAGCTG-3′; P3, 5′-GCAAGCTTATGGCGGCTCTGAAGAGTTG-3′; P4, 5′-GCGGATCCTCCTCACGCAGGTAGGCCT-3′; P5, 5′-CGGTCGACGTTAATTCTTCAAACTGCTT-3′; P6, 5′-GCGATTAATATGGCGGTTCCTATTGCAC-3′; P7, 5′-CGGCGGCCGCATCCTCACGCAGGTAGGCCT-3′; P8, 5′-CGGGATCCGATTAATATGAGGAGAGCAGTGTCTTT-3′; P9, 5′-CGGGATCCTGATGGCGGTTCCTATTGCACAG-3′; P10, 5′-GGGAGCCAGATCGCGACCCCATAGAGGA-3′; P11, 5′-CTATGGGGTCGCGATCTGGCTCCCAGCC-3′; and P12, 5′-CGGGATCCTCAATCCATGGCAGCCAGCT-3′.Reagents and Antibodies—The following antibodies were used in this study: polyclonal antibodies Ab-caspase-7, Ab-caspase-3, Ab-survivin, and Ab-actin (Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal antibodies: mAb-bcl2 (Oncogene, Manhasset, NY), mAb-GFP (MBL, Japan), mAb-cytochrome c (R&D Systems, Inc., Minneapolis, MN), mAb-caspase-9 (Immunotech, France), and mAb-Smac/DIABLO (Calbiochem, La Jolla, CA). Taxol used in our experiments is labeled as GCP grade. Restriction enzymes were mostly purchased from New England BioLabs. Medium compounds were obtained from Oxid. The majority of biochemical reagents were ordered from Sigma. Trypan blue was purchased from Invitrogen.Cell Culture and Transfection—HeLa cells were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, 1 × nonessential amino acid, 1 × minimal essential medium sodium pyruvate, 100 μg/ml penicillin, 100 μg/ml streptomycin (Invitrogen, Grand Island, NY). Cultured cells were incubated in a humidified atmosphere containing 5% CO2 at 37 °C. Transfection of cells with various mammalian expression constructs by LipofectAMINE 2000 (Invitrogen) was carried out according to the methods provided by the manufacturer's instructions.PCR-mediated Mutagenesis—To obtain the mutant survivin, we have employed the PCR-mediated mutagenesis method. Two pairs of primers, P1/P10 (complementing the regions from nucleotides 1 to 18 and nucleotides 196 to 224, respectively) and P11/P2 (complementing the regions from nucleotides 200 to 228 and nucleotides 408 to 426, respectively), were used to amplify two Survivin fragments. After gel purification, the resultant two overlapping PCR fragments were mixed with equal amounts. This mixture was incubated first at 94 °C for 4 min, followed by first PCR (PCR1) of 94 °C denaturing for 1 min, 56 °C annealing for 1 min, and 72 °C extension for 1 min. After 10 cycles, another 8-min extension was added to ensure the completion of all the extension at 72 °C. The PCR1 products were used as a template in a second PCR (PCR2), primed by oligonucleotides P10 and P11 to carry out another 20 cycles of PCR with the same PCR conditions used in PCR1. A prominent band with an expected size of 0.42 kb was visible on 1% agarose gel. A point mutation, GAC (Asp) to CGC (Arg), was further verified by DNA sequencing determination.Plasmids Construction—The full-length Survivin coding sequence (nucleotides 1–426) and its mutant variant with a point mutation at amino acid residue 71 (D71R) were cloned into BamHI/SalI sites of pGEX-5X-3 (Amersham Biosciences, UK), respectively, to generate recombinant expression vectors pGEX-5X-3/Survivin and pGEX-5X-3/Surv-D71R, and both fragments were inserted in-frame with the GST gene. The gene fragment (nucleotides 1–291), containing only the Survivin BIR domain (residues 1–97), was generated by the PCR method using primers P1 and P5 (see "Oligonucleotides") and cloned into BamHI/SalI sites of pGEX-5X-3 to yield pGEX-5X-3/Surv-BIR. Gene fragments coding for wild type Survivin, for mutant Survivin (Surv-D71R) with a point mutation at amino acid residue 71, for the truncated Survivin (Surv-BIR) lacking its C-terminal and Smac/DIABLO were also cloned into pEGFP-C1 plasmid, respectively. In addition, the gene fragments (reacted by PCR for primer pairs P12/P1) was used to generated pTRE2/A-Surv (coding for antisense Survivin). The DNA fragment coding for mature Smac/DIABLO (residues 56–239) and DNA fragment coding for mutant Smac/DIABLO (Δ74Smac, residues75–239) were generated by PCR techniques using primer pairs P6/P7 and P8/P7, respectively. The resultant PCR products were digested with restriction enzymes VspI and NotI and cloned into NdeI/NotI sites of pET-22b (Novagen, Madison, WI) to generate pET-22b/Smac and pET-22b/Δ74Smac with an His6 tag fusion at its C terminus. A full-length Smac/DIABLO gene fragment (residues1–239) resulted from PCR amplification with primers P3 and P4 inserted into HindIII/BamHI sites of the mammalian expression vector pEGFP-N1 (Clontech, Palo Alto, CA) to fuse to the EGFP gene, and the resultant recombinant plasmid was designated as pEGFP-N1/F-Smac. DNA fragments, coding for mutant Smac/DIABLO with an methionine residue added at the N terminus of mature Smac/DIABLO (designated as M-Smac) and for mutant Smac/DIABLO lacking 73 amino acid residues at its N terminus (designated as Δ73Smac), were reacted by PCR away from Smac/DIABLO cDNA using primer pairs P9/P4 and P8/P4, respectively, and inserted into the BamHI site of pEGFP-N1 in fusion with EGFP to generate plasmids pEGFP-N1/M-Smac and pEGFP-N1/Δ73Smac.Expression and Purification of Fusion Proteins—Overnight cultured Escherichia coli cells DH5α or BL21/DE3 containing the fusion plasmids were grown at a 1:100 dilution in an Amp-LB medium until the optical density reached A600 = 0.5. Isopropyl-1-thio-β-d-galactopyranoside was then added to a final concentration of 0.4 mm. Cells were induced for another 3 h before they were harvested and subjected to sonication for 10 shot pulses of 20 s each. The maximum protein release was determined by the Bradford assay (Bio-Rad) by comparing the known concentration of bovine serum albumin (BSA). The GST fusion proteins were purified through the glutathione-Sepharose™ 4B beads (Amersham Biosciences). The solubilized His6-Smac or His6-Δ74Smac fusion proteins were purified by incubating with chelating Sepharose™ Fast Flow beads (Amersham Biosciences) according to the manufacturer's instructions. The ultimate elution products were dialyzed with 1× PBS and were used for in vitro interaction assay.In Vitro Interaction Assay—An in vitro interaction assay was performed as described by Suzuki et al. (50Shin S. Sung B.J. Cho Y.S. Kim H.J. Ha N.C. Hwang J.I. Chung C.W. Jung Y.K. Oh B.H. Biochemistry. 2001; 40: 1117-1123Crossref PubMed Scopus (677) Google Scholar). Taxol-stimulated HeLa cells (2 × 106) were lysed with the 200-μl lysis buffer as described above. Cell lysates or bacterially expressed and purified His6 tag proteins were incubated with GST or GST fusion protein immobilized on glutathione-Sepharose 4B beads (Amersham Biosciences) for overnight at 4 °C and washed three times with 500 μl of 1× PBS. The bound proteins were eluted by elution buffer, and eluted products were subjected to SDS-PAGE for Western blot analysis or Coomassie Blue staining.Cell Death Assay—The ability of Survivin, Smac/DIABLO, or their mutants to affect cell viability was assayed by transfecting or HeLa cells (2 × 104 cells/well) in 24-well plates with 0.3 μg of mammalian expression vectors using LipofectAMINE 2000. Twenty hours after transfection, cells continued to be incubated with 100 nm Taxol for 36 h, and the viability of the cells was measured with the standard trypan blue exclusion method by counting blue dead cells. Data are expressed as percentages of control and are the means of three independent experiments.Western Blot Analysis—Cells were washed with 1× PBS and resuspended with 5 volumes of cold lysis buffer (50 mm Tris-HCl (pH 7.5), 250 mm NaCl, 5 mm EDTA, 50 mm NaF, 0.5% Nonidet P-40) supplemented with protease inhibitor mixture (Roche Applied Science). The cell lysate was incubated on ice for 30 min and was then centrifuged for 10 min at 4 °C. The protein content of the supernatant was determined by using a BCA-200 protein assay kit (Pierce). Equal amounts of proteins (10–20 μg) were loaded onto the gel and separated by SDS-PAGE, and the resolved proteins were transferred to nitrocellulose membrane. After blocking with 5% nonfat milk in TBST (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% Tween 20) for overnight at 4 °C, the blot was incubated with primary antibody for1hat room temperature. The membrane was then washed with TBST and probed with horseradish peroxidase-conjugated secondary antibody for 1 h. The membrane was washed three times in TBST and developed by ECL using the manufacturer's protocol. Digitonin fractionation of cells into membrane and cytosolic fractions used for detection of cytochrome c and Smac/DIABLO was performed as described by Ekert et al. (23Ekert P.G. Silke J. Hawkins C.J. Verhagen A.M. Vaux D.L. J. Cell Biol. 2001; 152: 483-490Crossref PubMed Scopus (162) Google Scholar).Immunoprecipitation—Cells were lysed in a Triton X-100-based lysis buffer (1% Triton X-100, 10% glycerol, 150 mm NaCl, 20 mm Tris, pH 7.5, 2 mm EDTA, protease inhibitor mixture) for 1 h, and the nuclear and cellular debris was cleared by centrifugation. Then the cytosolic lysis was incubated with Smac monoclonal antibody bound to Protein A/G-Sepharose. After a 4-h incubation at 4 °C, the immunoprecipitates were washed five times in lysis buffer, and proteins were recovered by boiling the beads in SDS sample buffer and analyzed using a Western blot.Immunofluorescence Confocal Microscopy—HeLa cells were grown in six-well chamber slides, and 24 h later, cells were either left untreated or exposed to 100 nm Taxol for another 24-h incubation before 100 nm MitoTracker Red was added and treated for 60 min at 37 °C. The cells were then washed with PBS, fixed with 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and blocked for 1 h in 3% BSA in PBS at 4 °C. Cells were incubated with anti-Smac (1:400 in 3% BSA) or anti-cyto c antibody (1:400 in 3% BSA) for 1 h at room temperature. The primary antibody was recognized by secondary FITC-conjugated antibody. Finally, the stained cells were analyzed with a laser scanning confocal microscopy system (Fluoview, Olympus).RESULTSSmac/DIABLO Is Released from the Mitochondria Accompanied with Cytochrome c upon Induction of Apoptosis by Taxol— Taxol is a natural product with potent anti-tumor activity and has been approved for the treatment of breast, ovarian, and lung cancers (25Marone M. D'Andrilli G. Das N. Ferlini C. Chatterjee S. Scambia G. Exp. Cell Res. 2001; 270: 1-12Crossref PubMed Scopus (31) Google Scholar, 30Rowinsky E.K. Cazenave L.A. Donehower R.C. J. Natl. Cancer. Inst. 1990; 82: 1247-1259Crossref PubMed Scopus (1099) Google Scholar, 31Panvichian R. Orth K. Day M.L. Day K.C. Pilat M.J. Pienta K.J. Cancer Res. 1998; 58: 4667-4672PubMed Google Scholar, 32Nicolini G. Rigolio R. Miloso M. Bertelli A.A. Tredici G. Neurosci. Lett. 2001; 302: 41-44Crossref PubMed Scopus (79) Google Scholar). We incubated HeLa cells with Taxol at a concentration of 100 nm, which is known to induce apoptosis in a number of different cell types. After 24-h incubation, the clear morphological changes characteristic of apoptosis were observed. Taxol-treated cells became rounded and detached from the substratum of the flask, followed by cellular shrinkage and membrane blebbing (data not shown). Caspase activation was believed to be the key factor during apoptosis. To evaluate which of the caspases is activated during the process of Taxol-induced apoptosis in HeLa cells, we analyzed the cleavage pattern of caspase-9, caspase-7, and caspase-3 by Western analysis methods. The results, shown in Fig. 1A, indicate that treatment with this drug results in the activation of caspase-9, caspase-7, and caspase-3 as evidenced by the appearance of caspase-active forms. One apoptotic signaling pathway leading into caspase activation involves the translocation of cytochrome c into the cytosol from the mitochondrial intermembrane space (33Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4435) Google Scholar). To further investigate whether the mitochondria are involved in this event, Taxol-treated HeLa cells were first lysed and divided into membrane pellet and cytosolic fraction. The pellet was then treated with 0.5% bile acid deoxycholate and separated by centrifugation into a soluble and membrane fraction. Cytosolic proteins and membrane proteins solubilized by the deoxycholate treatment were subjected to Western blot analysis. As shown in Fig. 1B (panel a), an increasing amount of cytochrome c was detected in the cytosolic fractions, whereas a decreasing amount of cytochrome c remained in the mitochondrial membrane, and after 48 h, the majority of cytochrome c was detected in the cytosolic fractions. We also observed that Smac/DIABLO was predominantly present within the membrane fractions prior to treatment with Taxol, and by 48 h post-treatment, there was a significant loss of Smac/DIABLO from the membrane fractions and a concomitant increase of Smac/DIABLO in the cytosolic fractions (Fig. 1B, panel a). In addition, to further elucidate the behavior of Smac/DIABLO or cyto c during Taxol-induced apoptosis, we examined its subcellular localization in both untreated cells and treated cells by immunostaining and confocal microscopy (Fig. 1B, panel b). HeLa cells were stained with MitoTracker Red to define the location of mitochondria and with anti-Smac or anti-cyto c antibody plus an FITC-conjugated secondary antibody. In uninduced HeLa cells, the localization of FITC and MitoTracker Red staining gave rise to the yellow color indicating that both Smac/DIABLO and cyto c were localized within the mitochondria. However, after induction with Taxol for 24 h, both Smac/DIABLO and cyto c were partially released into cytoplasm from mitochondria evidenced by their changed localization pattern different from MitoTracker Red staining. Thus we have demonstrated that Taxol treatment results in the release of cyto c and Smac/DIABLO, suggesting that the mitochondria pathway is involved.Ectopic Overexpression of Smac/DIABLO Sensitizes HeLa Cells to Taxol-induced Apoptosis—Smac/DIABLO is capable of promoting apoptosis induced by various apoptotic stimuli, including TRAIL, UV irradiation, and etoposide (22Chauhan D. Hideshima T. Rosen S. Reed J.C. Kharbanda S. Anderson K.C. J. Biol. Chem. 2001; 276: 24453-24456Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 23Ekert P.G. Silke J. Hawkins C.J. Verhagen A.M. Vaux D.L. J. Cell Biol. 2001; 152: 483-490Crossref PubMed Scopus (162) Google Scholar). We have demonstrated that treatment of Taxol could result in the release of Smac/DIABLO from mitochondria (Fig. 1B); it is therefore rational to propose that released cytosolic Smac/DIABLO may increase the susceptibility of HeLa cells to Taxol induction. To test this hypothesis, we transiently transfected HeLa cells with plasmids pEGFP-N1/F-Smac, pEGFP-N1/M-Smac, pEGFP-N1/Δ73Smac, and pEGFP-N1 separately (Fig. 2A). After 24 h of transfection, cell lysates were immunoblotted with anti-GFP antibody. Fig. 2B shows that full-length Smac (F-Smac) and its deleted mutants (M-Smac and Δ73Smac) were overexpressed in the transfected cells but not in the control cells transfected with vector. Mature Smac resulted from F-Smac cleavage from mitochondria upon Taxol induction was also examined by Western blot (Fig. 2B, lane 3). We then determined whether overexpressed Smac/DIABLO promoted apoptosis induced by Taxol. 20 h after transfection, cells were treated with 100 nm Taxol and incubated for another 36 h then collected and subjected to trypan blue staining. As shown in Fig. 2C, elevated expression of full-length Smac, which was cleaved after mitochondria import into the mature Smac/DIABLO, significantly increased Taxol-induced apoptosis, whereas two Smac-deleted mutants, M-Smac and Δ73Smac, which were reported to be unable to bind to XIAP (26Wu G. Chai J. Suber T.L. Wu J.W. Du C. Wang X. Shi Y. Nature. 2000; 408: 1008-1012Crossref PubMed Scopus (702) Google Scholar, 27Srinivasula S.M. Datta P. Fan X.J. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 28Srinivasula S.M. 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