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• W1988759802 abstract "MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce ∼1.4-MDa and ∼700-kDa apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This ∼700-kDa apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the apoptosome and forms a stable ∼200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the apoptosome and also forms a stable ∼200-kDa complex with active caspase-7. MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce ∼1.4-MDa and ∼700-kDa apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This ∼700-kDa apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the apoptosome and forms a stable ∼200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the apoptosome and also forms a stable ∼200-kDa complex with active caspase-7. The MCF-7 cell line was derived from a patient with metastatic breast cancer and is an often-used model system for studying estrogen receptor-positive breast cancer (for review, see Ref. 1.Simstein R. Burow M. Parker A. Weldon C. Beckman B. Exp. Biol. Med. 2003; 228: 995-1003Crossref PubMed Scopus (200) Google Scholar). Because many of the problems associated with breast cancer treatment involve the development of chemo-resistance to apoptosis-inducing anti-cancer agents, there is extensive interest in using MCF-7 cells as a model for investigating the mechanisms of apoptosis in breast epithelial cells. Caspase activation is a key event in triggering the morphological and biochemical changes associated with cell death (2.Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar, 3.Hengartner M. Science. 1998; 281: 1298-1299Crossref PubMed Google Scholar, 4.Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2450) Google Scholar). There are two primary caspase activation pathways involving either stimulation of cell surface death receptors (the extrinsic pathway) or perturbation of mitochondria (the intrinsic pathway) (5.Bratton S.B. MacFarlane M. Cain K. Cohen G.M. Exp. Cell Res. 2000; 256: 27-33Crossref PubMed Scopus (291) Google Scholar). Many anti-cancer drugs induce apoptosis by activating the intrinsic cell death pathway, which involves the release of cytochrome c and the activation of the apoptosome-catalyzed caspase cascade (6.Adrain C. Martin S.J. Trends Biochem. Sci. 2001; 26: 390-397Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 7.Cain K. Bratton S.B. Cohen G.M. Biochimie (Paris). 2002; 84: 203-214Crossref PubMed Scopus (359) Google Scholar, 8.Cain K. Drug Metab. Rev. 2003; 35: 337-363Crossref PubMed Scopus (66) Google Scholar). In apoptotic cells inactive procaspases are activated via this cascade mechanism in which an initiator caspase is activated and subsequently cleaves/activates an effector caspase, which then cleaves and activates the next caspase and so on. Caspase-3 is the most active effector caspase in both the intrinsic and extrinsic pathways, where it is processed and activated by caspase-9 and caspase-8, respectively. However, MCF-7 cells do not possess caspase-3 due to a 47-base pair deletion in the caspase-3 gene (9.Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar, 10.Kurokawa H. Nishio K. Fukumoto H. Tomonari A. Suzuki T. Saijo N. Oncol. Rep. 1999; 6: 33-37PubMed Google Scholar). Consequently, apoptotic cell death in MCF-7 cells must be independent of caspase-3 activation, although several studies have shown that apoptotic cell death in MCF-7 cells is accompanied by caspase activation. For example, in staurosporine-treated MCF-7 cells, recognized caspase death substrates including poly(ADP-ribose) polymerase, Rb, PAK2, gelsolin, and DFF-45 are cleaved (11.Janicke R.U. Ng P. Sprengart M.L. Porter A.G. J. Biol. Chem. 1998; 273: 15540-15545Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). In TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis, caspase-8 is activated, which cleaves Bid to release tBid, which in turn induces cytochrome c release and caspase-9 and caspase-7 processing (12.MacFarlane M. Merrison W. Dinsdale D. Cohen G.M. J. Cell Biol. 2000; 148: 1239-1254Crossref PubMed Scopus (150) Google Scholar). Other studies have also shown that caspases 9, 6, 2, and 7 are cleaved/processed to their active forms (13.Tang D. Lahti J.M. Kidd V.J. J. Biol. Chem. 2000; 275: 9303-9307Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Thus, in the absence of caspase-3, MCF-7 cells can still activate a caspase cascade irrespective of whether the apoptosis is initiated via the intrinsic or extrinsic pathway. The intrinsic pathway, which is activated by many chemicals, including anti-cancer drugs, involves formation of the Apaf-1 apoptosome, a large caspase-processing complex (for review, see Refs. 6.Adrain C. Martin S.J. Trends Biochem. Sci. 2001; 26: 390-397Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar and 7.Cain K. Bratton S.B. Cohen G.M. Biochimie (Paris). 2002; 84: 203-214Crossref PubMed Scopus (359) Google Scholar) that typically activates caspase-3. Apoptosome formation can be modeled in vitro in cell-free lysates by the addition of dATP or ATP and requires at least three apoptotic protease-activating factors (Apaf-1–3) (14.Zou H. Henzel W.J. Liu X.S. Lutschg A. Wang X.D. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar, 15.Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar, 16.Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6259) Google Scholar). The CARD (caspase recruitment) domain of Apaf-1 binds to a similar domain on procaspase-9, whereas the central CED-4 domain (98–412) is involved in Apaf-1 oligomerization. In the presence of cytochrome c and dATP, Apaf-1 undergoes conformational changes, allowing it to oligomerize to form a very large apoptosome complex. Using gel filtration chromatography, we have isolated from both dATP-activated THP.1 and B chronic lymphocytic leukemia cell lysates two apoptosome complexes with apparent molecular masses of ∼700 kDa and ∼1.4 MDa (17.Cain K. Brown D.G. Langlais C. Cohen G.M. J. Biol. Chem. 1999; 274: 22686-22692Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 18.Cain K. Bratton S.B. Langlais C. Walker G. Brown D.G. Sun X.M. Cohen G.M. J. Biol. Chem. 2000; 275: 6067-6070Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 19.Almond J. Snowden R.T. Dinsdale D. Hunter A. Cain K. Cohen G.M. Leukemia. 2001; 15: 1388-1397Crossref PubMed Scopus (79) Google Scholar). Furthermore, we have shown in apoptotic cells that the ∼700-kDa complex predominates and is the most active complex in processing exogenous procaspase-3 (18.Cain K. Bratton S.B. Langlais C. Walker G. Brown D.G. Sun X.M. Cohen G.M. J. Biol. Chem. 2000; 275: 6067-6070Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 19.Almond J. Snowden R.T. Dinsdale D. Hunter A. Cain K. Cohen G.M. Leukemia. 2001; 15: 1388-1397Crossref PubMed Scopus (79) Google Scholar). More recently, we have used a proteomic approach to characterize the composition of this ∼700-kDa apoptosome complex and have shown that it contains solely Apaf-1 and caspase-9 as its core functional proteins (20.Twiddy D. Brown D.G. Adrain C. Jukes R. Martin S.J. Cohen G.M. MacFarlane M.M. Cain K. J. Biol. Chem. 2004; 279: 19665-19682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Several studies have reported that caspase-7 is cleaved and activated in dATP stimulated lysates (17.Cain K. Brown D.G. Langlais C. Cohen G.M. J. Biol. Chem. 1999; 274: 22686-22692Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 21.Slee E.A. 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. Martin S.J. J. Cell Biol. 1999; 144: 281-292Crossref PubMed Scopus (1686) Google Scholar, 22.Bratton S.B. Walker G. Srinivasula S.M. Sun X.M. Butterworth M. Alnemri E.S. Cohen G.M. EMBO J. 2001; 20: 998-1009Crossref PubMed Scopus (342) Google Scholar), and immuno-depletion studies indicate that caspase-9 is required for the processing of both caspase-3 and -7 (21.Slee E.A. 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. Martin S.J. J. Cell Biol. 1999; 144: 281-292Crossref PubMed Scopus (1686) Google Scholar). By implication, it has been assumed that the Apaf-1-caspase-9 apoptosome complex directly processes procaspase-7 in an analogous manner to that described for procaspase-3. However, there is no direct evidence for this, and to address this, we have characterized the role of the apoptosome in caspase-7 activation in MCF-7 cells. Our studies show that the ∼700-kDa apoptosome complex is also formed in dATP-activated MCF-7 cell lysates and apoptotic MCF-7 cells and directly cleaves and activates procaspase-7. However, although the kinetics of Apaf-1 oligomerization is normal, the recruitment and processing of caspase-9 in the holoenzyme complex is much slower than in caspase-3-containing cell lysates. We also show that caspase-7, after activation by the apoptosome complex, forms a XIAP-caspase-7 complex that is not disrupted even in the presence of Smac and Omi/HtrA2. These data suggest that the absence of caspase-3 enables XIAP to have an enhanced inhibitory effect on effector caspase activation and activity and, hence, a more significant anti-apoptotic role in MCF-7 cells. Cell Culture, Apoptosis Assays, and Preparation of Control and Caspase-activated Cell Lysates—MCF-7- and MCF-7-transfected cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 2 mm Glutamax™ as described previously (12.MacFarlane M. Merrison W. Dinsdale D. Cohen G.M. J. Cell Biol. 2000; 148: 1239-1254Crossref PubMed Scopus (150) Google Scholar). For apoptosis assays, cells were harvested by mild tryptic digestion, and the percentage of apoptotic cells with exposed phosphatidylserine was determined by annexin V-fluorescein isothiocyanate binding (Bender Medsystems, Vienna, Austria) and fluorescence-activated cell sorter essentially as previously described (12.MacFarlane M. Merrison W. Dinsdale D. Cohen G.M. J. Cell Biol. 2000; 148: 1239-1254Crossref PubMed Scopus (150) Google Scholar). Loss of mitochondrial membrane potential (ΔΨm) was measured with the lipophilic cationic fluorescent probe tetramethylrhodamine ethyl ester and a fluorescence-activated cell sorter scan analysis (23.Dewson G. Snowden R.T. Almond J.B. Dyer M.J. Cohen G.M. Oncogene. 2003; 22: 2643-2654Crossref PubMed Scopus (98) Google Scholar). For assaying cytochrome c release, cells were resuspended in cold phosphate-buffered saline and permeabilized with 0.025% digitonin, the cell cytosol and membrane fractions were prepared by centrifugation, and the cytochrome c content of the various fractions was then determined by SDS-PAGE and Western blotting (24.Sun X.M. MacFarlane M. Zhuang J.G. Wolf B.B. Green D.R. Cohen G.M. J. Biol. Chem. 1999; 274: 5053-5060Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). Cell lysates (100,000 × g supernatants) from cells were prepared by freeze/thawing (F/T) 2The abbreviations used are: F/T, freeze/thawing; AFC, 7-amino-4-trifluoromethylcoumarin; CHAPS, 3-([3-cholamidoopropyl]-dimethylammonio)-1-propanesulfonate); IAP, inhibitor of apoptosis; XIAP, X-linked IAP; IBM, IAP binding motif; Dig/Hom, digitonin and homogenization; casp, caspase; WT, wild type; fmk, fluoromethyl ketone. (7.Cain K. Bratton S.B. Cohen G.M. Biochimie (Paris). 2002; 84: 203-214Crossref PubMed Scopus (359) Google Scholar, 18.Cain K. Bratton S.B. Langlais C. Walker G. Brown D.G. Sun X.M. Cohen G.M. J. Biol. Chem. 2000; 275: 6067-6070Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). In some experiments, to minimize mitochondrial breakage and release of pro-apoptotic proteins, MCF-7 cell lysates (digitonin and homogenization (Dig/Hom)) were prepared in an isotonic buffer (MSH), containing 210 mm mannitol, 70 mm sucrose, 5 mm Hepes, 1 mm EGTA, 1 mg/ml Pefabloc SCD, one protease inhibitor mixture tablet/10 ml (Roche Diagnostics), pH 7.4, using a modified digitonin/homogenization technique (20.Twiddy D. Brown D.G. Adrain C. Jukes R. Martin S.J. Cohen G.M. MacFarlane M.M. Cain K. J. Biol. Chem. 2004; 279: 19665-19682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Caspase activation in lysates (10–15 mg/ml) was induced by incubation at 37 °C for various times with 2 mm dATP/MgCl2 plus or minus 2.0 μm cytochrome c. Preparation of Recombinant Procapases 3 and 7—BL21 (DE3) cells were transformed with a pET-21b (Novagen) plasmid expression vector containing procaspase-3 or -7 cDNA with a C-terminal His6 purification tag and grown at 37 °C in terrific broth, 1% glucose, and ampicillin (100 μg/ml) essentially as described previously (20.Twiddy D. Brown D.G. Adrain C. Jukes R. Martin S.J. Cohen G.M. MacFarlane M.M. Cain K. J. Biol. Chem. 2004; 279: 19665-19682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Fluorimetric Assays of Caspase and Caspase-activating Activity— DEVDase activity (i.e. primarily caspase-3 and -7) of lysates or column fractions was measured fluorimetrically with 200 μl of assay buffer (20 μm Ac-DEVD.AFC, 0.1% CHAPS, 10 mm dithiothreitol, 100 mm HEPES, and 10% sucrose, pH 7.0) using a Wallac Victor 2The abbreviations used are: F/T, freeze/thawing; AFC, 7-amino-4-trifluoromethylcoumarin; CHAPS, 3-([3-cholamidoopropyl]-dimethylammonio)-1-propanesulfonate); IAP, inhibitor of apoptosis; XIAP, X-linked IAP; IBM, IAP binding motif; Dig/Hom, digitonin and homogenization; casp, caspase; WT, wild type; fmk, fluoromethyl ketone. 1420 Multilabel counter (18.Cain K. Bratton S.B. Langlais C. Walker G. Brown D.G. Sun X.M. Cohen G.M. J. Biol. Chem. 2000; 275: 6067-6070Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). In some experiments the Ac-DEVD.AFC substrate concentration was increased to 100 μm. The caspase processing/activating activity of soluble apoptosome complexes was assayed using purified recombinant procaspase-3 or procaspase-7 (20.Twiddy D. Brown D.G. Adrain C. Jukes R. Martin S.J. Cohen G.M. MacFarlane M.M. Cain K. J. Biol. Chem. 2004; 279: 19665-19682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Briefly, the method assays the DEVDase activity of the sample, which is a direct measure of how much procaspase-3/-7 has been processed and activated by the apoptosome. In addition, aliquots of the DEVD assay mixtures were diluted 1:1 with 2× SDS loading buffer and analyzed for caspase-3/-7 processing by SDS-PAGE and Western blotting. Fractionation of Cell Lysates by Gel Filtration—Lysates were fractionated by size-exclusion chromatography on either Superose-6 or Sephacryl S300 columns using a fast protein liquid chromatography (HR 10/30 column) protein purification system (Amersham Biosciences). Columns were eluted at 4 °C with 5% (w/v) sucrose, 0.1% (w/v) CHAPS, 20 mm HEPES/NaOH, 5 mm dithiothreitol, and 50 mm NaCl, pH 7.0, calibrated with protein standards as previously described (18.Cain K. Bratton S.B. Langlais C. Walker G. Brown D.G. Sun X.M. Cohen G.M. J. Biol. Chem. 2000; 275: 6067-6070Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Column fractions (0.5–2 ml) were analyzed for DEVDase activity and caspase-3 or -7 processing activity as described above. Aliquots of the column fractions were also analyzed by SDS-PAGE and Western blotting for Apaf-1, caspases 9, 7, and 3 and XIAP. For the Western blot analysis, column fractions 5–17 and 18–30 were run on separate gels, and immunoblotting procedures were carried out in parallel. To ensure uniformity in signal response, the two blots from each column run were exposed simultaneously to Kodak X-Omat film. Appropriate fractions were pooled and concentrated with Vivaspin concentrators (Vivascience AG, Hannover, Germany) before immunoprecipitation experiments with the indicated antibodies. Affinity Purification of Caspase-7 Complexes—Immunopurification of caspase-7 complexes was carried out using either an anti-active caspase-7 (Cell Signaling, New England Biolabs UK Ltd., Hertfordshire, UK) or anti-XIAP (clone 48, BD Biosciences Pharmingen) antibody covalently bound, respectively, to protein A and G Dyna Beads® (20.Twiddy D. Brown D.G. Adrain C. Jukes R. Martin S.J. Cohen G.M. MacFarlane M.M. Cain K. J. Biol. Chem. 2004; 279: 19665-19682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Immunopurification of caspase complexes was carried out from MCF-7 cell lysates (∼30 mg/ml) that had first been precleared by incubating (1 h at 4 °C) with 400 μl of Sepharose protein A-coated beads/ml of lysate. The precleared lysates were then diluted to 15 mg/ml with assay buffer and dATP-activated for 2 h at 37 °C before fractionation by gel filtration. Appropriate fractions containing active caspase-7 complexes were pooled and concentrated before adding antibody-tagged beads and roller mixing at 4 °C for 4 h. Affinity purified proteins were eluted from the beads with SDS-PAGE sample loading buffer and separated by one-dimensional SDS-PAGE. In some experiments apoptosome complexes were affinity-purified using an anti-caspase-9 antibody covalently attached to protein G Dyna Beads® (20.Twiddy D. Brown D.G. Adrain C. Jukes R. Martin S.J. Cohen G.M. MacFarlane M.M. Cain K. J. Biol. Chem. 2004; 279: 19665-19682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Apoptosome complexes were captured by adding 20 μl of cross-linked beads to 90–100 μl of dATP-activated MCF-7 cell lysate and roller mixing overnight at 4 °C. Proteins were eluted from the beads as described above. Reagents and Western Blot Analysis—Cell culture media and materials were as previously described (12.MacFarlane M. Merrison W. Dinsdale D. Cohen G.M. J. Cell Biol. 2000; 148: 1239-1254Crossref PubMed Scopus (150) Google Scholar, 25.Sun X.M. Butterworth M. MacFarlane M. Dubiel W. Ciechanover A. Cohen G.M. Mol. Cell. 2004; 14: 81-93Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Most other reagents and cells, unless indicated otherwise, were obtained from published sources (20.Twiddy D. Brown D.G. Adrain C. Jukes R. Martin S.J. Cohen G.M. MacFarlane M.M. Cain K. J. Biol. Chem. 2004; 279: 19665-19682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). MCF-7 cells stably transfected with caspase-3 (MCF-7/casp-3) or pcDNA3 vector only (MCF-7/vector) cells were a gift from Dr. Alan Porter, (National University of Singapore) and originally characterized elsewhere (9.Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar). Column, cell lysates, and immunopurified samples were analyzed for various proteins by SDS-PAGE and Western blotting as previously described (20.Twiddy D. Brown D.G. Adrain C. Jukes R. Martin S.J. Cohen G.M. MacFarlane M.M. Cain K. J. Biol. Chem. 2004; 279: 19665-19682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). An antibody to procaspase-7 was obtained from BD Biosciences Pharmingen. Antibodies to active caspase-3 and -7 were sourced from Cell Signaling. Caspase Activation in dATP/Cytochrome c-treated MCF-7 Cell-free Lysates Does Not Require Caspase-3—In this study we wished to investigate apoptosome formation and function in MCF-7 cells. It was, therefore, important to characterize cytochrome c and dATP-dependent caspase activation in the cell lysates. This in vitro model has been used with a number of cell lines, and caspase-3-containing THP.1 cell lysates undergo a marked increase (∼30–50-fold) in DEVDase activity (largely due to caspase-3 processing) when activated with dATP/cytochrome c (17.Cain K. Brown D.G. Langlais C. Cohen G.M. J. Biol. Chem. 1999; 274: 22686-22692Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). However, dATP/cytochrome c treatment of MCF-7 lysates produced only a very small increase in DEVDase activity (Fig. 1A) even though SDS-PAGE/Western blot analysis showed that caspase-9 was processed to its p35/p34 forms and caspase-7 was cleaved to its p19 active form (lane 3, Fig. 1A). In contrast, MCF-7 cell lysates, activated with dATP and then incubated with recombinant procaspase-3, exhibited a marked increase in DEVDase activity that was accompanied by procaspase-3 processing to its p19 and p17 forms (lane 6, Fig. 1A). Caspase-3 markedly influenced the processing of procaspase-9, which was initially cleaved to the p37 (caspase-3 dependent) and p35 forms when the cell lysate was activated by dATP alone (lane 5, Fig. 1A) and then fully processed primarily to the p37 and p35 forms in the presence of dATP/cytochrome c (lane 6, Fig. 1A). Also, caspase-3 accelerated the processing of procaspase-7 to its fully cleaved p19 active form (lane 6, Fig. 1A). Interestingly, in the presence of recombinant procaspase-3, the p32 form of caspase-7 was detected when the cell lysate was activated with dATP alone (lane 5, Fig. 1A). This agrees with previous studies showing that the p32 subunit is formed by caspase-3 cleavage of procaspase-7, which removes the prodomain at Asp-23 (26.Denault J.B. Salvesen G.S. J. Biol. Chem. 2003; 278: 34042-34050Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The modest increase in DEVDase activity observed in the dATP/cytochrome c-treated MCF-7 lysates could possibly be explained by the fact that caspase-7 has a higher Km and a lower kcat value for the DEVD synthetic peptide substrate (26.Denault J.B. Salvesen G.S. J. Biol. Chem. 2003; 278: 34042-34050Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 27.MacFarlane M. Cain K. Sun X.M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar, 28.GarciaCalvo M. Peterson E.P. Rasper D.M. Vaillancourt J.P. Zamboni R. Nicholson D.W. Thornberry N.A. Cell Death Differ. 1999; 6: 362-369Crossref PubMed Scopus (195) Google Scholar). The results also suggested that caspase activation in MCF-7 cell lysates was slower than in caspase-3-containing lysates. We investigated this by comparing dATP-dependent caspase activation in MCF-7 cells stably transfected with either pcDNA3 (vector) or pcDNA-casp-3. Although in MCF-7/vector cell lysates the p35 subunit was detected 5 min after dATP/cytochrome c activation, processing of the procaspase-9 was not complete until 120 min (lane 7, Fig. 1B). The p34 subunit was not detected until 60 min after dATP/cytochrome c activation, whereas in MCF-7/casp-3 cell lysates, procaspase-9 cleavage was accelerated and almost complete after 10 min (lane 12, Fig. 1B). Thus, the p35 subunit was detected at 5 min, reached a maximum at 30 min, and remained constant until 120 min before declining to barely detectable levels by 360 min. The p37 subunit was detected after the appearance of the p35 subunit at 10 min and was maximal after 30 min before disappearing by 60–120 min. The time course also showed that caspase-7 activation in MCF-7/vector cell lysates was subsequent to caspase-9 processing, as the p19 subunit of caspase-7 was not detected until 30 min after treating with dATP/cytochrome c (lane 5, Fig. 1B). By this time there was already significant processing of procaspase-9 to its p35 form. The p22 subunit of caspase-7 (see 15-min exposure, Fig. 1B) was also detected, indicating that removal of the prodomain to produce the fully processed p19 form does not require caspase-3. In contrast, in caspase-3-containing cell lysates, caspase-7 processing was faster and more extensive than in the MCF-7/vector cell lysates and was paralleled by rapid and extensive processing of caspase-3 to the p20, p19, and p17 forms. In MCF-7 Cell Lysates, Apaf-1 Oligomerizes to Form Two Apoptosome Complexes—We next established that Apaf-1 could form a fully functional apoptosome complex in MCF-7 cell lysates. Gel-filtration chromatography of control lysates showed that Apaf-1 eluted as an ∼130-kDa monomer (fraction 18–21, Fig. 2A). After dATP/cytochrome c activation, most of the Apaf-1 eluted either as ∼700-kDa (fraction 9–14, Fig. 2A) or ∼1.4-MDa (fraction 5–8, Fig. 2A) apoptosome complexes. In control cell lysates procaspase-9 (∼46-kDa) eluted in fractions 20–24 (Fig. 2A). After dATP activation, both the proform and processed (p35) form eluted in fractions 20–24 (Fig. 2A). However, processed caspase-9 was not associated with either the ∼700-kDa or ∼1.4-MDa apoptosome complexes. This contrasts with dATP-activated THP.1 cell lysates, where processed caspase-9 was detected in both apoptosome complexes and also as the free form (18.Cain K. Bratton S.B. Langlais C. Walker G. Brown D.G. Sun X.M. Cohen G.M. J. Biol. Chem. 2000; 275: 6067-6070Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Next, we immunoblotted the column fractions for caspase-7, and in MCF-7/WT control lysates procaspase-7 eluted predominantly in fractions 20–24 (Fig. 2A), corresponding to a molecular mass of ∼60–80 kDa. However, after dATP activation, the active p19 form of caspase-7 eluted in fractions 18–21, which corresponds to a molecular mass of ∼200–300 kDa (Fig. 2A). The ∼200–300-kDa caspase-7 complex we detected in MCF-7/WT cell lysates did not contain Apaf-1 or caspase-9 (Fig. 2A). To determine whether or not the elution pattern of caspase-7 was different from that of caspase-3, we also fractionated MCF-7/casp-3 lysates by Superose-6 gel filtration chromatography. In these lysates Apaf-1 and caspase-9 exhibited similar profiles of oligomerization and cleavage, respectively, as did the MCF-7/WT lysates (Fig. 2B). Procaspase-3 eluted in fractions 21–24 (Fig. 2B) and is slightly smaller than procaspase-7. However, in dATP/cytochrome c-treated lysates, the active p19/17 forms of caspase-3 eluted in fractions 22–26 and not in the caspase-7-containing fractions (Fig. 2B). This is consistent with the known quaternary structure of active caspase-3, which is a 58–60-kDa tetramer and indicated that active caspase-7 forms a larger ∼200-kDa complex with one or more proteins. The ∼700-kDa Apoptosome Complex Directly Processes Procaspase-7—We next investigated the ability of the ∼700-kDa and ∼1.4-MDa apoptosome complexes to directly activate effector caspases. Column fractions were incubated with procaspase-3, which is cleaved/activated by the Apaf-1-caspase-9 apoptosome. Caspase activation was determined by measuring DEVDase activity, which was predominant in fractions 9–14 (Fig. 3A). These fractions corresponded to the ∼700-kDa apoptosome complex, as determined by their Apaf-1 content (Fig. 2A). Those fractions corresponding to the ∼1.4-MDa apoptosome complex did not activate procaspase-3, and column fractions taken from fractionated non-activated cell lysates also did not activate this procaspase (Fig. 3A). From this we concluded that the ∼700-kDa complex was the only active apoptosome complex. The fact that it processed and activated procaspase-3 indicated that it should also contain caspase-9. However, we were unable to detect caspase-9 in the column fractions (Fig. 2A), as possibly the concentration of apoptosome was" @default.
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• W1988759802 title "Caspase-7 Is Directly Activated by the ∼700-kDa Apoptosome Complex and Is Released as a Stable XIAP-Caspase-7 ∼200-kDa Complex" @default.
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