Báo cáo khoa học: a-Fetoprotein positively regulates cytochrome c-mediated caspase activation and apoptosome complex formation docx

a-Fetoprotein positively regulates cytochrome c -mediated caspase activation and apoptosome complex formation Lidia Semenkova 1, *, Elena Dudich 1, *, Igor Dudich 1 , Natalie Tokhtamisheva 1 , Edward Tatulov 2 , Yury Okruzhnov 3 , Jesus Garcia-Foncillas 3 , Juan-Antonio Palop-Cubillo 4 and Timo Korpela 5 1 Institute of Immunological Engineering, Moscow, Russia; 2 Anticancer Drug Research Center, Moscow, Russia; Departments of 3 Oncology and 4 Organic Chemistry and Pharmacology, University of Navarra, Pamplona, Spain; 5 Joint Finnish-Russian Biotechnology Laboratory, Turku University, Finland Previous results have shown that the oncoembryonic marker a-fetoprotein (AFP) is able to induce apoptosis in tumor cells through activation of caspase 3, bypassing Fas- dependent and tumor necrosis factor receptor-dependent signaling. In this study we further investigate the molecular interactions involved in the AFP-mediated signaling of apoptosis. We show that AFP treatment of tumor cells is accompanied by cytosolic translocation of mitochondrial cytochrome c. In a cell-free system, AFP mediates process- ing and activation of caspases 3 and 9 by synergistic enhancement of the low-dose cytochrome c-mediated sig- nals. AFP was unable to regulate activity of caspase 3 in cell extracts depleted of cytochrome c or caspase 9. Using high-resolution chromatography, we show that AFP posit- ively regulates cytochrome c/dATP-mediated apoptosome complex formation, enhances recruitment of caspases and Apaf-1 into the complex, and stimulates release of the active caspases 3 and 9 from the apoptosome. By using a direct protein–protein interaction assay, we show that pure human AFP almost completely disrupts the association between processed caspases 3 and 9 and the cellular inhibitor of apoptosis protein (cIAP-2), demonstrating its release from the complex. Our data suggest that AFP may regulate cell death by displacing cIAP-2 from the apoptosome, resulting in promotion of caspase 3 activation and its release from the complex. Keywords: apoptosis; apoptosome; cytochrome c;IAP-2; a-fetoprotein. Apoptotic cell death is characterized by biochemical and morphological changes, which are largely caused by caspase activity. A class of cysteine proteases, known as caspases, which are constitutively expressed in cells as inactive proenzymes, require proteolytic cleavage to be activated. In general, either receptor-induced or mitochondrion- induced death signals stimulate activation of specific adapterproteinsFADD/MORT1orApaf-1byformation of the high-molecular-mass death-inducing complex or apoptosome. The adapter proteins recruit initiator caspases 8 and 9 to activate them by autoprocessing. Once activated, initiator caspases are ready to induce processing of down- stream effector caspases 3 and 7 [1]. The mitochondrial apoptosis pathway is mediated by cytochrome c (cyt-c) release with the subsequent formation of the Apaf-1/cyt-c/ dATP/procaspase 9 apoptosome complex, leading to acti- vation of caspase 9 and downstream effector caspases [2]. Chromatographic analysis of the apoptosome assembly indicated that, in native cell lysates, Apaf-1 oligomerizes into multimeric complexes of molecular mass  1.4 MDa and  700 kDa, which in addition to processed caspase 9, contain fully processed caspase 3 and 7 [3]. Caspases are inhibited by a number of cellular inhibitor of apoptosis proteins (cIAPs), which bind directly to procaspases 9 and 3 to prevent their cyt-c-mediated processing and activation [4,5]. During apoptosis, a mitochondrial protein named Smac/DIABLO [6] that directly binds to IAPs to remove them from the apoptosome complex [4,7], cancels the IAP-mediated caspase inhibition. Recently, another IAP- inhibitory protein Omi/HtrA2 was characterized, which operates by abrogation of the IAP–caspase interaction [8]. AFP is the major serum protein of embryonic plasma that is involved in regulation of gene expression, differen- tiation, proliferation and apoptosis in developing cells [9–12]. Although, the biological role of this protein is not yet fully understood, it has been well characterized as a physiological carrier/transport protein for various ligands, including fatty acids, drugs, hormones, heavy metals, delivering them to developing and malignant cells [9,12]. The specific expression and internalization of AFP is restricted to developing cells, such as embryonic cells, activated immune cells and tumor cells, which suggests its important regulatory role in cell growth and differentiation [9,10,12]. Various researchers have documented the exist- ence of specific receptor-dependent mechanisms responsible for the active endocytosis of AFP by malignant cells [13,14]. Microscopic data have demonstrated that fluoresceinated Correspondence to E. Dudich, Institute of Immunological Engineering, 142380, Lyubuchany, Moscow Region, Chekhov District, Russia. Tel./Fax: + 7 095 996 15 55, E-mail: dudich@ineos.ac.ru Abbreviations: AFP, a-fetoprotein; cyt-c, cytochrome c; cIAP, cellular inhibitor of apoptosis protein; Ac-DEVD-AMC, Ac-Asp-Glu-Val- Asp-7-amino-4-methylcoumarin; LEHD-AFC, Leu-Glu-His-Asp- aminotrifluoromethylcoumarin; IETD-AMC, Ile-Glu-Thr-Asp-7- amino-4-methylcoumarin; CHO, aldehyde. *Note: These authors contributed equally to this work. (Received 11 February 2003, revised 28 August 2003, accepted 16 September 2003) Eur. J. Biochem. 270, 4388–4399 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03836.x AFP is specifically bound to the cell surface at 4 °Cand internalized into the cytoplasm at 37 °C [15,16]. It has been shown that AFP is internalized via coated pits and vesicles before being delivered to endosomes [15,16]. Much evidence of cell growth regulatory activity, including tumor suppres- sion, has been reported for various species of the full-length AFP molecule [17–22], its proteolytic fragments [23], recombinant domains [24] and synthetic peptides [25–27]. It has been demonstrated that AFP realizes its tumor- suppressive activity by triggering apoptosis, characterized by typical morphological changes, growth arrest, cytotoxi- city, and DNA fragmentation [20–22]. It was shown that AFP induces apoptosis in malignant cells through activa- tion of caspase 3, bypassing Fas/FasL and tumor necrosis factor (TNF)/TNFR-dependent pathways and does not require upstream activation of receptor-dependent initiatory caspase 8 and caspase 1 [21]. Although these studies have shown that a caspase cascade is initiated during AFP- induced apoptosis, the mechanisms by which AFP triggers caspase activation are unknown. Our previous experimental data show that AFP does not require de novo protein synthesis and RNA expression to trigger apoptosis, as it was not blocked by actinomycin D or cycloheximide [20]. In this study, we aimed to determine how AFP activates the caspase cascade. To understand the molecular mecha- nisms of AFP-mediated apoptosis signaling, we established a cell-free system, similar to that used for studies of cyt-c- induced apoptosis [28,29]. We show here that AFP syner- gistically enhances caspase activation and processing in the presence of a low suboptimal dose of cyt-c and requires the presence of all members of the apoptosome complex to initiate this process. We examine the mechanisms by which AFP regulates apoptosis and demonstrate that the pro- apoptotic effect of AFP is mediated through its interaction with apoptosome-forming proteins. Chromatographic ana- lysis of the apoptosome assembly demonstrated that AFP stimulates formation of the Apaf-1–apoptosome complex, enhances recruitment and activation of procaspase 3 in the complex, and stimulates release of active caspase 3 and 9 from the apoptosome. Our data suggest that AFP may regulate cell death by displacing cIAP-2 from the apopto- some complex, thereby promoting caspase 3 release from the complex. Materials and methods AFP purification Human AFP was isolated from the cord serum using ion- exchange, affinity and gel-filtration chromatography as described previously [23]. AFP purity was established using PAGE and immunoblotting with monospecific antibodies against human AFP and adult serum proteins and was showntobenolessthan99.8%. Cells HepG2 cells originated from the American Type Culture Collection were cultured in Dulbecco modified Eagle’s medium (ICN Biomedicals) with L -glutamine and 10% heat-inactivated fetal bovine serum, 100 IU penicillinÆmL )1 , 0.1 mg streptomycinÆmL )1 in a humidified 5% (v/v) atmosphere of CO 2 at 37 °C. For a passage, cells were incubated in 0.25% (v/v) trypsin solution, then washed and plated out. Cytotoxicity assay HepG2 cells were incubated with 5–7 l M AFP for deter- mined time intervals of 2–14 h, and then assessed for their viability by the trypan blue exclusion assay as described previously [22]. Cells cultivated without additions were taken as a control. The experimental data were expressed as the percentage of dead cells relative to the total amount of cells. Preparation of cell-free extracts Cell-free S-100 extracts were generated from human hepatocarcinoma HepG2 as described [29,30]. Cells (4 · 10 8 ) were collected and washed (three times) in 50 mL NaCl/P i and once in 5 mL hypotonic cell extraction buffer (containing 20 m M Hepes, pH 7.2, 10 m M KCl, 2m M MgCl 2 ,1m M dithiothreitol, 5 m M EGTA, 25 lgÆmL )1 leupeptin, 5 lgÆmL )1 pepstatin, 40 m M b-glycero- phosphate, 1 m M phenylmethanesulfonyl fluoride). The cell pellet was then resuspended in an equal volume of cell extraction buffer, allowed to swell for 20 min on ice, and then disrupted by 30–50 strokes of a Dounce homogenizer. The homogenate was centrifuged at 3000 g for 10 min at 4 °C to remove whole cells and nuclei. The supernatant was centrifuged at 15 000 g for 20 min at 4 °Candthen,to obtain the cytosolic S-100 extract, the supernatant was re-centrifuged at 100 000 g for 1 h at 4 °C. Extracts were assessed for protein content by the Bradford assay and stored in aliquots at )70 °C. Cyt c-free cytosolic extracts were prepared in more mild conditions by the slightly modified procedure described in [30]. In vitro caspase activation For in vitro caspase activation, 40 lg of the S-100 extract (complete or after immunodepletion) was incubated for the indicated times with bovine heart cyt-c (Sigma-Aldrich, St Louis, MO, USA) and/or pure human AFP (5 l M )inthe presence or absence of 1 m M dATP (Sigma) in 15 lLofa reaction buffer (10 m M Hepes, pH 7.2, 25 m M NaCl, 2 m M MgCl 2 ,5m M dithiothreitol, 5 m M EDTA, 0.1 m M phenyl- methanesulfonyl fluoride) at 30 °C. To control specificity of AFP effects, the equivalent amount of human serum albumin (Sigma) was added instead of AFP. The activity and proteolytic processing of caspases 3 and 9 were then detected by fluorimetric assay and immunoblotting with the corres- ponding antibodies supplied by Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA): polyclonal goat anti-(caspase 3) p20 (N19); anti-(caspase 3) p11 (K19); rabbit anti- (caspase 9) p10 (H-83); rabbit anti-(caspase 9) p35 (H-170). Fluorimetric assay of caspase activity Caspase activities were determined by incubation of the extract aliquots (5 lL) for various times at 30 °C with one of the fluorogenic substrates [40 l M Ac-DEVD-AMC (ICN Biomedicals Inc), 50 l M LEHD-AFC (Chemicon Ó FEBS 2003 AFP amplifies cytochrome c-mediated caspase activation (Eur. J. Biochem. 270) 4389 International, Temecula, CA, USA) or 50 l M IETD-AMC (Alexis Biochemicals, San Diego, USA] in 16 lL substrate buffer (25 m M Hepes, pH 7.2, 100 m M NaCl, 1 m M EDTA, 0.1% Chaps, 10 m M dithiothreitol, 10% sucrose). Reactions were terminated by dilution with 2.0 mL ice-cold 0.2 m M sodium phosphate buffer, pH 7.5, and fluorescence was measured using a Perkin–Elmer MPF-44A fluorimeter (k exc ¼ 365 nm and k em ¼ 440 nm for the AMC fluores- cence or k exc ¼ 400 nm and k em ¼ 505 nm for the AFC fluorescence). For each sample, caspase activity was expressed in relative units, pmolÆmin )1 Æmg )1 , showing the amount of cleaved substrate in pmol normalized for time of reaction with substrates and cytosolic protein concentra- tion, or in relative fluorescent units (FU) per fraction. Immunoprecipitation and immunoblotting analysis S-100 cytosolic extracts obtained from HepG2 cells were immunodepleted from endogenous cyt-c, procaspase 9 or procaspase 3 by immunoprecipitation with the corres- ponding antibodies as described [31]. Briefly, 50 lLofthe S-100 cell extract (4–5 mgÆmL )1 ; reaction buffer with addition of 0.1% Chaps) was incubated for 2 h at 4 °C with 5 lg of the corresponding antibodies: anti-cyt-c 6H2.B4 (PharMingen, San Diego, CA, USA), anti- (caspase 9) clones C-18 and H-83 or anti-(caspase 3) (N-19). The control cell extracts were incubated with the equivalent amounts of the control antibodies of the same type. Immune complexes were precipitated by addition of antibody/extract mixture on to drained protein G-Seph- arose or protein A/agarose beads (Amersham Pharmacia Biotech) for 2 h at 4 °C. Coated beads were then removed by centrifugation, and the resulting immuno- depleted lysates after adjustment for protein concentration were used immediately for caspase activation experiments. The extent of depletion was controlled by immunoblot- ting with the corresponding antibodies. Immunoblotting with b-actin antibodies (ICN Biomedicals Inc) was performed as a loading control. For immunoblotting analysis, protein samples (50 lgper lane) were subjected to standard SDS/PAGE in a 12% or 15% polyacrylamide gel and transferred on to 0.45-l M poly(vinylidene difluoride) membranes by semidry electro- blotting, followed by probing for various proteins using the corresponding antibodies: rabbit anti-(Apaf-1), H-324 (Santa Cruz); affinity-purified rabbit anti-(human cIAP-2), HIAP-1 (R & D Systems, Wiesbaden, Germany); rabbit polyclonal anti-(caspase 8) p20, H-134 (Santa Cruz) or the corresponding polyclonal antibody goat anti-(caspase 3) or anti-(caspase 9). Bound antibodies were detected using appropriate horseradish peroxidase-conjugated anti-rabbit or anti-goat secondary IgGs (Santa Cruz) and developed by enhanced chemiluminescence staining using ECL reagents (Amersham Pharmacia Biotech). Gel calibration was per- formed with the Low Molecular Weight Calibration Kit for SDS Electrophoresis (Amersham Pharmacia Biotech). Dot-blot analysis was performed as usual. Briefly, 1-lL aliquots taken from the chromatographic fractions were applied to the nitrocellulose membranes, then blocked by defatted milk. The membranes were then probed with rabbit polyclonal affinity-purified anti-(human AFP) IgG. Bound antibodies were detected using appropriate peroxidase- coupled secondary antibodies and developed as described above. Assay of cyt-c release Cyt-c translocation from mitochondria to the cytoplasm was assessed by direct immunochemical measurement of the cyt-c in the cytosolic and mitochondrial fractions obtained from HepG2 cells treated with AFP for various time intervals. Briefly, cells (0.5 · 10 6 cells per well) in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum were plated on the flat-bottomed 24-well plates (Nunc) and incubated for 24 h. Then 5 l M AFP was added to each well. After various lengths of treatment (2–17 h), cells were scraped, washed in NaCl/P i , and resuspended in 200 lL digitonin lysis buffer (0.025% digitonin in 250 m M sucrose, 20 m M Hepes, pH 7.4, 5 m M MgCl 2 ,10m M KCl, 1 m M EDTA, 1 m M EGTA, 10 m M Tris/HCl, pH 7.4, 10 lgÆmL )1 leupeptin, 10 lgÆmL )1 aprotinin, and 1 m M phenylmethanesulfonyl fluoride) [32]. After 10 min, cell lysates were centrifuged for 2 min at 14 000 g at 4 °Cto obtain the supernatant (cytosolic fraction) and the pellet (mitochondrial fraction). Mitochondrial pellet was solubi- lized by a 30-min incubation with 100 lL lysing buffer (150 m M NaCl, 1% Nonidet P40, 0.5% deoxycholate, 0.1% SDS, 50 m M Tris/HCl, pH 7.5, cocktail of protease inhi- bitors). Thereafter, cellular debris was removed by a 10-min centrifugation at 14 000 g at 4 °C. The supernatant com- prising the membrane fraction was retained. Equal amounts of cytosolic extracts and solubilized mitochondrial pellets (50 lg protein) were fractionated by SDS/PAGE using 15% polyacrylamide and then analysed by Western blot using the cyt-c antibody 7H8.2C12, cyt-c oxidase subunit II antibody (Molecular Probes), and b-actin antibody and ECL as described above. Direct protein–protein interaction assay To determine possible interactions between AFP and caspase 3, caspase 9 and cIAP-2, we used a direct copre- cipitation assay with purified proteins. Before the experi- ments, 25 lL Ni/Sepharose beads (Qiagen, Valencia, CA, USA) were incubated for 1 h at 20 °C in a solution of assay buffer (50 m M Tris/HCl, 100 m M KCl, 10% sucrose, 0.1% Chaps, 0.5 m M dithiothreitol, pH 7.4), containing 1% ovalbumin, 12 lg His-tagged human recombinant caspase 9 and 3 lg active His-tagged rat recombinant caspase 3 (Alexis Biochemicals). After being washed, one half of the beads was added to the cytosolic extract of HepG2 cells (500 lg total protein) together with 20 lgAFP andincubatedfor2hat4°C. The control beads were incubated with the same amount of HepG2 cytosolic extract without AFP addition. The protein–bead complexes were then washed (four times), isolated by centrifugation, boiled in 15 lL sample buffer, and analyzed by SDS/PAGE/ Western blotting with anti-cIAP2 (HIAP-1) IgG. Chromatographic analysis of the apoptosome assembly To study effects of AFP on recruitment, processing and release of various caspases from apoptosome and micro- apoptosome complexes, we used the previously described 4390 L. Semenkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003 gel filtration technique [3]. Briefly, S-100 extracts were prepared from HepG2 cells (6 mgÆmL )1 ) and activated by a 1-h incubation at 30 °C with 1.0 m M dATP/1.5 m M MgCl 2 / 1.0 l M cyt-c with or without 5.0 l M AFP. Before addition to the S-100 extracts, AFP samples were dialyzed against the elution buffer. Activated lysate proteins ( 1mg) were applied (0.2 mLÆmin )1 ;4°C) to a 10/30 Superose-6 HR column connected to an FPLC system (Amersham Phar- macia Biotech). The column was eluted with elution buffer (20 m M Hepes/KOH, 10 m M KCL, 1 m M EDTA, 1 m M EGTA, 1 m M dithiothreitol, 1.5 m M MgCl 2 ,0.01m M phenylmethanesulfonyl fluoride, pH 7.2); 1-mL fractions were collected. Aliquots of the fractions were taken for measurement of caspase activity using the corresponding fluorogenic substrates: DEVD-AMC for caspase 3 and Ac- IETD-AMC for caspases 9 and 8 [33] as described above. Fractions were then concentrated 20-fold with 2 mL centrifugal concentrators (Centricon YM-10; Amicon) and analyzed by PAGE and immunoblotting for changes in distribution of AFP, Apaf-1, cIAP-2, caspases 3, 9 and 8. Column calibration was performed with Gel Filtration LMW and HMW calibration kits (Amersham Pharmacia Biotech). Results AFP induces release of mitochondrial cyt-c in HepG2 cells Our previous publications were devoted to the study of AFP-induced apoptosis in whole cells and suggested that this mechanism is independent of membrane receptor signaling [20–23]. We investigate here the intracellular molecular pathways of the AFP-mediated triggering of apoptosis. To analyse the involvement of cyt-c release in AFP-mediated apoptosis, cytosolic and mitochondrial fractions were obtained from AFP-treated HepG2 cells and analysed by Western blot for the presence of cyt-c. As shown in Fig. 1, AFP induced the appearance of cyt-c in the cytosolic fraction of treated HepG2 cells and its disappear- ance from the mitochondrial fraction of treated cells, indicating that AFP induced mitochondrial cyt-c release. These data do not show, however, whether AFP induces cytosolic cyt-c release directly or by indirect mechanisms by activation of unknown factors. AFP synergistically enhances low-dose cyt-c-mediated caspase activation in cell-free cytosolic extracts The mitochondrial apoptotic pathway could be activated by addition of dATP to cell extracts to initiate the Apaf-1/ procaspase 9/cyt-c apoptosome cascade [28]. To determine whether AFP is involved in this process, we established a typical cell-free system using HepG2 cells and measured caspase activation in this system with or without addition of AFP. Two types of cell lysate were used for these experiments: a typical S-100 cytosolic extract and a cyt-c- free cytosolic extract, prepared by a mild procedure as described previously [30]. Addition of AFP to the S-100 cytosolic extract triggered dATP-dependent induction of caspase 3-specific DEVDase activity, which progressively increased for at least 2 h (Fig. 2A). As a control, the equivalent amount of human serum albumin was added to the same cell-free system. No effect was observed at the level of DEVDase activity. A low level of DEVDase activity was also induced by dATP alone, evidently due to the presence of a small amount of endogenous cyt-c in the preparations. In the absence of dATP, AFP did not induce any caspase 3- specific DEVDase activity at all. To determine whether AFP can directly induce caspase activation in cell-free cytosolic extract or requires the presence of the basal level of cyt-c, we examined DEVDase cleavage activity after addition of exogenous cyt-c and AFP to the ÔsilentÕ cytosolic extracts with undetectable endo- genous cyt-c. Figure 2B shows that no DEVDase activity was detected in this type of cytosolic lysate stimulated with dATP/AFP or with dATP and low suboptimal dose of cyt-c even 1.5 h after treatment. A significant time-dependent increase in DEVDase activity was observed in the same reaction system only after addition of all three compounds: AFP,dATPandcyt-c(Fig.2B).ThelowDEVDaseactivity in this experimental system compared with that described in Fig. 2A is explained by the negligible amount of cyt-c in the cytosol. These data demonstrate the ability of AFP to amplify caspase-activating signals induced by low subopti- mal doses of cyt-c. We then examined the effect of AFP on the DEVDase activity mediated by different doses of cyt-c in S-100 extracts. Figure 2C shows that, similarly to the above data (Fig. 2A), AFP synergistically enhances DEVDase activity induced by low suboptimal doses of cyt-c. A further increase in cyt-c concentration in the cell extract resulted in the ÔsaturationÕ effect, when the maximal stimulation of caspase 3-specific DEVDase activity was reached, which AFP cannot further increase (Fig. 2C). Fig. 1. Effect of AFP on cell viability and cyt-c release in HepG2 cells. HepG2 cells were treated with 5 l M AFP for various time intervals, and then cytosolic and mitochondrial extracts were prepared at the indicated times. Equal amounts of cytosolic and mitochondrial extracts (50 lg) were immunoblotted with anti-(cyt-c) to assess cyt-c release. b-Actin and cytochrome oxidase subunit II (Cyt ox.) were also analysed in cytosolic and mitochondrial extracts as controls for protein loading. Cell viability of AFP-treated HepG2 cells was assessed by the trypan blue exclusion assay as described in Materials and methods. Ó FEBS 2003 AFP amplifies cytochrome c-mediated caspase activation (Eur. J. Biochem. 270) 4391 AFP synergistically enhances cyt-c-mediated processing and activation of procaspases 9 and 3 in cell-free cytosolic extracts To determine whether AFP could induce increased caspase activation in a cell-free system, we examined S-100 extracts for cleavage of procaspases 3 and 9 and corresponding fluorogenic caspase substrates after addition of AFP/cyt-c/ dATP. Both procaspase 9 and procaspase 3 were processed to their active forms, giving the corresponding fragments p35/37 and p10 for caspase 9 and p17 and p12 for caspase 3. However, when AFP was combined with cyt-c/ dATP, more complete cleavage of the procaspases was observed (Fig. 3B,C). In addition, there was a dramatic increase in caspase 3-like DEVDase activity and a notable increase in caspase 9-like LEHDase activity on combined treatment with AFP/cyt-c/dATP in comparison with cyt-c/ dATP (Fig. 3A). These data show that AFP positively regulates both processing and activation of procaspases 9 and 3 in cell-free cytosolic extracts by amplification of the low-dose cyt-c-mediated effects. AFP induces caspase activation only in the presence of the all components of the apoptosome complex The above experiments demonstrated functional interfer- ence of AFP with the cyt-c-mediated process of caspase activation. We studied further the functional significance of Fig. 2. AFP enhances cyt-c-mediated DEVDase activity in cell-free cytosolic extracts. (A) AFP induces caspase 3 activation in cell-free S-100 cytosolic extracts in the presence of dATP. Effect of endogenous cyt-c. Aliquots of HepG2-derived cytosolic extract (25 lgprotein) were treated for various times with AFP (5 l M ) or as a control with the same dose of human serum albumin in the presence of dATP (1 m M ) and then assayed for DEVDase activity. (B) Synergistic increase in DEVDase activity mediated by AFP in cyt-c-free cytosolic extracts on addition of exogenous cyt-c. Aliquots of the cyt-c-free HepG2-derived cytosolic extracts (25 lg protein) were treated for various times with AFP (5 l M ), cyt-c (0.2 l M ) or a combination of the same doses of the two compounds in the presence of dATP (1 m M ) and then assayed for DEVDase activity. (C) AFP differently affects caspase 3 activation in cell-free cytosolic extracts induced by various doses of cyt-c. Aliquots of S-100 cytosolic extract (25 lg protein) were treated for 30 min with AFP (5 l M ) and various doses of cyt-c in the presence of dATP (1 m M ) and then assayed for DEVDase activity. The mean ± SD from four determinations is shown. Fig. 3. AFP positively regulates cyt-c-mediated DEVDase and LEH- Dase activity and processing of procaspase 9 and 3 in a cell-free system. Aliquots of HepG2-derived S-100 cytosolic extract with addition of 1m M dATP were treated in the presence (+) or absence (–) of cyt-c (0.2 l M ) and/or AFP (5 l M ). (A) Proteolytic activities of caspase 9 and 3 in experimental lysates were assayed by monitoring the cleavage of the corresponding fluorogenic substrates LEHD-AFC and Ac-DEVD- AMC. The mean ± SD from four determinations is shown. Processing of caspases was detected by immunoblotting with the cor- responding antibodies that recognize the precursors and subunits of active caspase 9 (B) and 3 (C). 4392 L. Semenkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003 AFP in regulation of activity of the apoptosome complex. Cellular extracts were sequentially depleted of the main active molecular compounds involved in the formation of the apoptosome complex: endogenous cyt-c, procaspase 3, or procaspase 9. Caspase activation was then induced by the addition of cyt-c/dATP with or without AFP. AFP was unable to induce caspase 3 activation in the absence of cyt-c and/or dATP in the cyt-c-immunodepleted cytosolic extracts (Fig. 4). However, addition of exogenous cyt-c together with dATP produced DEVDase activity. Simultaneous addition of all three compounds (AFP, cyt-c and dATP) resulted in significant enhancement of total DEVDase activity com- pared with that induced with cyt-c/dATP (Fig. 4). We next determined whether AFP requires the presence of procaspase 9 to induce caspase 3 activation mediated by a suboptimal dose of cyt-c. HepG2 S-100 extracts were depleted of procaspase 9 by immunoprecipitation with the corresponding antibody and then treated with AFP/cyt-c or cyt-c alone in the presence of dATP. Figure 5A,B shows that removal of caspase 9 from cell extracts led to the complete loss of AFP/cyt-c-mediated DEVDase activity, whereas control extracts and extracts treated with anti-RXR (antibody control) displayed significant enhancement of the total cyt-c-mediated DEVDase activity in response to AFP addition. These results are supported by additional data showing that the specific caspase 9 inhibitor Ac-LEHD- CHO significantly suppressed cyt-c/dATP-dependent AFP- mediated DEVDase activity in a cell-free system (Fig. 5B). The results show that AFP with or without cyt-c cannot directly induce caspase 3 activation in a cell-free system in the absence of procaspase 9. AFP cannot induce activation of procaspase 9 in the absence of caspase 3 As AFP was unable to activate procaspase 3 in the absence of procaspase 9, we further studied whether AFP is capable of activating procaspase 9 independently of caspase 3. HepG2-derived S-100 cytosolic extracts were depleted of procaspase 3 by immunodepletion with the corresponding Fig. 4. Depletion of cyt-c abrogates AFP-mediated caspase activation in cytosolic extracts. (A) Endogenous cyt-c was removed from S-100 cytosolic extract by immunoprecipitation with anti-cyt-c mAb 6H2.B4. To confirm cyt-c depletion, equal amounts (50 lg) of control untreated extract, extract treated with unspecific mouse IgG (antibody control) and cyt-c-depleted extract were resolved by SDS/PAGE and immu- noblotted with anti-(cyt-c). b-Actin was used as a loading control. (B) Caspase activation in cyt-c-depleted lysate was induced by treatment with appropriate doses of AFP (5 l M ) and/or cyt-c (0.2 l M )inthe presence of dATP (1 m M ). Caspase 3 activity was measured by monitoring cleavage of the fluorogenic substrate DEVD-AMC. The mean ± SD from four determinations is shown. Fig. 5. Procaspase 9 is required for AFP-mediated caspase 3 activation. (A) S-100 cytosolic extract was immunodepleted of procaspase 9 by immunoprecipitation with anti-(caspase 9). To confirm caspase 9 depletion, equal amounts (50 lg) of control untreated extract, cyt-c- treated extract, extract treated with anti-RXR (control for possible unspecific antibody-induced effects) and caspase 9-depleted extract were analysed by immunoblotting with anti-(caspase 9). b-Actin was used as a loading control. (B) Caspase 3 activation was induced in different types of experimental extract: caspase 9-depleted extract, complete extract, complete extract incubated with Ac-LEHD-CHO and extract treated with anti-RXR. Extracts were activated by addition (+) or in the absence (–) of appropriate doses of AFP (5 l M )and/or cyt-c (0.2 l M ) in the presence of dATP (1 m M ). Caspase 3 activity was measured by cleavage of the fluorogenic substrate DEVD-AMC. The mean ± SD from four determinations is shown. Ó FEBS 2003 AFP amplifies cytochrome c-mediated caspase activation (Eur. J. Biochem. 270) 4393 antibody. Depletion was controlled by immunoblotting (Fig. 6A) and direct measurement of the DEVDase activity (not shown). Thereafter procaspase 3-depleted S-100 extracts were tested for LEHDase activity upon treatment with AFP and/or cyt-c. Addition of cyt-c to caspase 3-depleted extracts induced a distinct increase in LEHDase activity, showing caspase 9 activation (Fig. 6B). These data indicate that cyt-c induced dose-dependent activation of caspase 9 in a caspase 3-independent manner, demonstra- ting the hierarchical advantage of caspase 9 in this process. In contrast, treatment of caspase 3-depleted extracts with AFP did not induce any enhancement of LEHDase activity compared with the effect of cyt-c alone (Fig. 6B), showing that the presence of procaspase 3 is critical for the realization of AFP-mediated pro-apoptotic activity. AFP positively regulates cyt-c-mediated apoptosome complex formation in a cell-free system and release of active caspases from the complex Our current data demonstrate that AFP requires the presence of all of the main members of the apoptosome complex (cyt-c, dATP, caspases 9 and 3) to induce caspase activation in a cell-free system. We reasoned that AFP may be involved in regulating the activity of the apoptosome complex. To test this hypothesis, we studied the formation of the apoptosome complex in cell-free extracts induced by cyt-c/dATP in the presence or absence of AFP by monitoring the distribution of caspase activity along the chromatography pattern. To evaluate caspase 8 and caspase 9 activation, we measured IETDase cleavage activity. Figure 7A shows that caspase 8 is completely absent from the position of the active  700-kDa complex (fractions 8–10) and was detected only in fractions 14–15, corresponding to the free form of the processed enzyme, as described previously [31,33]. Thus, in the absence of caspase 8 in the apoptosome complex, IETDase cleavage activity in this region may represent effects induced by active forms of caspase 9 [34]. The data obtained from measurement of LEHDase cleavage activity showed signi- ficantly lower fluorescent intensity and were difficult to interpret (not shown). Our data demonstrate that AFP did not induce any changes in IETDase activity in the position of the active 700-kDa complex (fractions 8–10), but DEVDase activity in this region was notably enhanced compared with the effect of cyt-c alone (Fig. 7A,B). The most significant AFP-mediated increase in DEVDase cleavage activity was observed at  70–60 kDa (fractions 15–17), corresponding to the free active caspase 3 (Fig. 7B). Figure 7A shows that integral IETDase activity at  90 kDa corresponding to free active caspases 9 and 8 (fractions 14–15) was also enhanced after AFP addition (Fig. 7A). The distribution of caspase 9 and caspase 3 precursors and mature forms distinctly correlates with the corres- ponding activity patterns (Fig. 7A,B). Caspase 9 was processed under these conditions and showed two peaks in the column for both experimental systems with and without addition of AFP. The main peak of caspase 9- specific material was located in fractions 9–10, whereas the second peak was at fractions 13–15. It should be mentioned that a smaller amount of the processed caspase 9 was also detected in fractions 6–7, correspond- ing to the biologically inactive  1.4-MDa apoptosome complex (not shown), similarly to previously reported data [3,35]. Our data confirmed results obtained by these authors [3,35] indicating that in spite of the presence of all of the members of the apoptosome complex (Apaf-1, cyt-c, caspase 9) in the  1.4-MDa apoptosome complex, it was unable to cleave IETD-like substrates, showing its inability to process effector caspases. In the absence of AFP, the precursor of caspase 9 was recovered mainly in the free form in fractions 14–15, demonstrating that a low suboptimal dose of cyt-c does not recruit all the available procaspase 9 for apoptosome formation. A small amount of processed caspase 9 was also found in this case in fraction 13 corresponding to a molecular mass of  160–180 kDa, indicating the formation of an intermediate complex (Fig. 7A, bottom). After treatment of S-100 with AFP/cyt-c/dATP, we observed a significant increase in the total amount of the processed caspase 9 in fractions 14–15, indicating that AFP stimulates both maturation of caspase 9 and its release from the complex. In the S-100 extract, which was stimulated with cyt-c/ dATP, both precursor and processed forms of caspase 3 Fig. 6. AFP cannot induce activation of procaspase 9 in the absence of caspase 3. (A) Procaspase 3 was immunodepleted from S-100 extracts by immunoprecipitation with anti-(caspase 3). To confirm immuno- depletion, 50 lg protein from control complete extract, extract treated with goat IgG (control for possible unspecific antibody-induced effects) and caspase 3-depleted extract were analyzed by immuno- blotting with anti-(caspase 3). b-Actin was used as a loading control. (B) Caspase 9 activation in caspase 3-depleted extracts was induced by addition of the appropriate doses of AFP (5 l M ), cyt-c (0.2 l M ), and dATP (1 m M ) and assessed by cleavage of the fluorogenic substrate LEHD-AFC. The mean ± SD from four determinations is shown. 4394 L. Semenkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003 were detected mainly in fractions 13–14 ( 160– 180 kDa), reflecting activity distribution (Fig. 7B). These data indicate that, at low cyt-c, caspase 3, like caspase 9, tends to form an intermediate  160–180-kDa complex or migrate together with other protein aggregates in this region. In the extracts stimulated with AFP/cyt-c/dATP, we revealed the precursor form of caspase 3 in fractions 13–14, whereas processed caspase 3 was recovered mainly in fractions 15–16, showing again that AFP stimulates release of the free active caspase 3 from the complex. We have also monitored the distribution of AFP along the chromatography pattern of the S-100 extracts after addition of AFP/cyt-c/dATP and found this 70-kDa protein in fractions corresponding to the high-molecular-mass complexes (Fig. 7B, bottom). Pure protein migrates at the position corresponding to its monomeric size compared with the molecular mass standard. This indicates that AFP may be involved in formation of the high-molecular-mass multimeric complexes with cytosolic proteins and may modulate protein–protein interactions within the complexes. Fig. 7. AFP positively regulates formation of Apaf-1 apoptosome in cell-free extracts and promotes caspase activation and release of caspase 3 and 9 from the complex. Aliquots (1 mL) of S-100 extracts obtained from nonapoptotic HepG2 cells were left untreated (control) or activated by a 1-h incubation at 30 °Cwith1m M dATP and 0.7 l M cyt-c in the presence or absence of 5.0 l M AFP. Subsequently, the extract aliquot (1 mg protein) was fractionated by high-resolution chromatography on a Superose-6 HR 10/30 column. Fractions of 1 mL were collected and aliquots of 50 lL were assayed fluorimetrically for IETDase (A) and DEVDase (B) activity. Caspase activity is given in arbitrary fluorescent units in the fraction per minute. Arrowheads at the top of the patterns indicate sizes of calibration protein standards and their elution positions from the Superose-6 column. Dot-blot analysis of the AFP distribution in the fractions for cell lysates treated with AFP/dATP/cyt-c is shown under the chromatographic pattern (B). The corresponding fractions were concentrated, and aliquots of 20 lL were also resolved by SDS/PAGE and immunoblotting for caspase 9, caspase 8 (A), caspase 3 (B), Apaf-1 (C) and anti-(cIAP-2) (D). The corresponding chromatography fraction numbers are indicated under the patterns. The central line marked with an asterisk shows the blot of cell lysate with addition of cyt-c/dATP before chromatography. (E) AFP displaces endogenous cIAP-2 from the complex with caspases 3 and 9. Recombinant His-tagged active caspase 9 and caspase 3 were immobilized on the Ni/Sepharose beads and incubated with HepG2 S-100 extract with or without 5 l M AFP. Ni/Sepharose-bound proteins were analyzed by SDS/PAGE/immunoblotting with polyclonal antibodies to cIAP-2. Ó FEBS 2003 AFP amplifies cytochrome c-mediated caspase activation (Eur. J. Biochem. 270) 4395 Effect of AFP on the distribution of Apaf-1 and cIAP-2 proteins along the chromatographic pattern of the apoptosome assembly To determine possible mechanisms of the AFP-mediated regulation of the apoptosome complex, we monitored the distribution of Apaf-1 along the chromatographic pattern of the apoptosome assembly, which was formed with and without AFP (Fig. 7C). In cell extracts stimulated with low cyt-c, Apaf-1 was recovered in two main peaks correspond- ing to fractions 6–8 and 13–15, demonstrating that a low suboptimal dose of cyt-c does not recruit all the available Apaf-1 into the functional apoptosome and tends to form the nonfunctional complex of molecular mass  1.4 MDa. In the presence of AFP, Apaf-1 specificity was significantly reduced in the biologically inactive  1.4-MDa complex (fractions 6–7) [3,35], but notably increased in the region of the  700-kDa apoptosome (fractions 8–10). These data indicate that, at low cyt-c, AFP positively modulates recruitment of Apaf-1 into the active  700-kDa apopto- some complex. Figure 7D shows that cIAP-2 distribution was not so clearly affected by AFP addition as observed in the case of Apaf-1. However, in the absence of AFP, full-length cIAP-2 was present in fractions 10–11, whereas fraction 9 mainly contained fragmented IAP-2-specific material (Fig. 7D). After the addition of AFP, the cIAP-2 specificity (including full-length protein and its fragments) was distinctly reduced in fractions 9–10 (Fig. 7D). The similar fragmentation pattern for cIAP-1 and cIAP-2 has been described previ- ously [36]. It was shown that fragmented cIAP-1 and cIAP-2 were more effective at protecting cells from apoptosis, whereas full-length proteins lacked protective activity. Removal of the RING domain by proteolysis restored the antiapoptotic activity [36]. It was also shown that cIAP-1 was cleaved in vitro by pure caspase 3, producing similar 52-kDa and 35-kDa fragments. Our data allow us to suggest that AFP may negatively regulate fragmentation of cIAP-2, thus modulating its antiapoptotic activity. Alternatively, AFP may stimulate release of active fragmented cIAP from the apoptosome. From our data we proposed the possible interaction of AFP with cIAP-2 and its partial removal from the apoptosome complex. To confirm this, we studied the direct interaction of AFP and cIAP-2 using a direct protein– protein interaction assay. Interaction between caspase 9, caspase 3, c-IAP-2 and AFP To study further the interaction between AFP, cIAP-2 and caspases 9 and 3, we precipitated pure recombinant active caspases 3 and 9 (His-tagged) with nickel resin and then incubated them with AFP and S-100 extract, as a source of cIAP-2. A similar reaction mixture was also prepared without AFP. The supernatants and pellets were probed with antibodies against cIAP-2. As IAPs interact directly with active caspases 3 and 9 [5], we speculated that AFP may physically interact with one of these proteins to displace cIAP-2 from the complex. Figure 7E shows that cIAP-2 binds processed recombinant caspase 3 and/or 9. Addition of the pure human AFP in the same reaction system almost completely disrupts the interaction between processed caspases and cIAP-2, demonstrating its release from the complex (Fig. 7E). Additional experiments with each protein member of the complex will be necessary to clarify the exact molecular interactions involved in this effect. Our data suggest that AFP may positively regulate the activity of the apoptosome by negative modulation of the cIAP-2 content, resulting in promotion of the release of active caspases 3 and 9 from the complex. Discussion There is increasing evidence that AFP may selectively induce activation of programmed cell death in tumor cells [17–23], showing its potential for cancer treatment [10]. Various researchers have documented the tumor-selective uptake of AFP by malignant cells [13–16], but the functional significance of this phenomenon has not been clarified. The exact molecular mechanisms of AFP-mediated apoptosis also remain unclear. The present data explain some details of the molecular interactions in this effect. In this study we have investigated the ability of AFP to directly activate the death program in a cell-free model of apoptosis. Release of cyt-c into the cytoplasm of AFP- treated cells suggests that a mitochondrion-dependent mechanism of apoptosis signaling is involved. However, these data do not exclude the possibility that another cyt-c- independent pathway of AFP-mediated signaling of apop- tosis is also involved in the sequential indirect induction of cyt-c release with the onset of its activity. We found here that AFP promotes low-dose cyt-c/dATP-mediated pro- cessing and activation of procaspases 9 and 3 in a cell-free system. These data show that AFP is directly involved in regulating the mechanisms of caspase cascade activation and suggest that it may be involved in regulating apopto- some complex formation. We have demonstrated further that AFP-mediated signaling of apoptosis requires the presence of all the major members of the apoptosome complex: cyt-c, dATP, caspases 9 and 3. To confirm that AFP is involved in regulating activity of the apoptosome complex, cell-free cytosolic extracts were activated in vitro by addition of cyt-c/dATP or AFP/cyt-c/dATP, and, after high-resolution gel filtration, the fractions from the column were analysed by Western blotting. Our data clearly show that AFP positively regulates cyt-c/dATP-mediated forma- tion of the active  700-kDa Apaf-1–apoptosome complex and stimulates release of the active caspase 3 from the complex. The key was the finding that AFP negatively regulates binding of cIAP-2 to active caspases 9 and 3. It remains to be seen if AFP associates with and inhibits interaction of other cIAPs with caspases, thus promoting caspase activation within the apoptosome complex. Our data suggest that AFP interacts with caspase 3, 9 and/or cIAP-2 in a similar manner to DIABLO/Smac or Omi/Htr [7,8], but the exact molecular determinants involved in these interactions remain to be determined. A similar effect of selective triggering of apoptosis in tumor cells was observed for multimeric forms of human a-lactalbumin, MAL [37,38]. It was shown that only oligomerized forms of this protein are capable of inducing apoptosis. Our recent data similarly showed that AFP requires concentration-dependent oligomerization to become apoptotically active [23]. The exact mechanism of 4396 L. Semenkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003 MAL function has not yet been established, but it was shown that MAL induces its proapoptotic effects by direct activation of the caspase cascade independently of the membrane-receptor signaling [38]. To prevent uncontrolled proliferation of rapidly growing tissues, such as developing immature immune cells, embry- onic cells or tumor cells, certain natural control mechanisms have to exist that select and direct developing cells toward maturation and prevent their neoplastic transformation. This study describes a naturally occurring protein, the expression of which is restricted by developing immature embryonic cells or cells undergoing malignant transforma- tion [9–12]. Proteins with quite mundane functions in healthy cells often behave very differently during cell suicide. The selective proapoptotic activity of AFP, targeting only neoplastic [17–23] and activated immune cells [9,10], indi- cates that it is a natural effector in a fetoembryonic defense system to prevent malignant transformation of developing cells. Our data allow us to propose that AFP helps cells to overcome their resistance to apoptosis by significant ampli- fication of the apoptotic signals induced by other factors, such as drugs and oxidative stress. AFP may help cells, which are resistant to apoptotic stimuli for any reason, to overcome their resistance, which is induced, for example, by overexpression of heat shock proteins, cIAPs or any other defects of apoptosome-dependent apoptotic pathways. Tumor cells are characterized by defects in expression of apoptosis-promoting proteins, such as Apaf-1 and p53, and simultaneous overexpression of the antiapoptotic proteins Hsp70, Bcl-2 and Bcl-x L , resulting in tumor-specific sup- pression of apoptosis and enhancement of the malignance and therapy resistance of tumors [39–43]. The existence of a high background level of antiapoptotic factors in the cytosol of tumor cells often leads to their resistance to apoptosis induced by weak stress stimuli. It has been demonstrated that high levels of AFP in maternal serum during pregnancy were associated with a low incidence of breast cancer [44,45]. It was proposed that AFP may delete immature breast tissue cells that show the first signs of neoplastic transfor- mation. Our results correlate with these data, indicating that AFP may function to remove transformed neoplastic cells by tumor-selective amplification of weak apoptotic signals. Of special interest in cancer research are tumor-specific factors that regulate apoptosis in tumor cells which function at a common part of the apoptotic signaling pathway and may cancel their resistance to apoptosis. There are several hypotheses that may help to explain these findings. The release of cyt-c from the mitochondria into the cytosol has been shown to be one of the earliest apoptotic events, which occurs before mitochondrial depolarization, caspase activa- tion and DNA fragmentation. It has been documented that, after apoptosis signaling, cyt-c is released from the mito- chondria within 5 min [46]. The extramitochondrial cyt-c has been shown to be a general apoptogen in cells with a functional caspase system [47]. On the other hand, recent papers have shown that cells can survive with reduced mitochondrial membrane potential and released cytosolic cyt-c given appropriate signals to suppress apoptosis [48,49]. It was observed that the amount of released cyt-c in K562 and CEM lines did not correlate with the extent of apoptosis in response to UV light, showing reduced caspase 3 activa- tion. The effect was explained by the reduced expression of Apaf-1 protein in resistant leukemic cells [48]. A high background level of cytosolic cyt-c has been shown in vivo in the aging heart, with a significant decrease in the antiapop- totic protein bcl-2 [49]. In certain types of cancer cell, alterations in the regulation of apoptosis may contribute to tumor malignancy and resistance to radiotherapy and chemotherapy [50]. Sometimes dysfunctional apoptosome activation in tumor cells is observed in the presence of the required amount of cytosolic cyt-c, dATP, Apaf-1 and pro- caspase 9, leading to significant enhancement of their resistance to apoptotic stimuli including radiotherapy and chemotherapy [51]. Our data indicate that AFP can be considered as a tumor- specific regulator of cyt-c-mediated apoptotic signals. In vivo, it may operate as a specific regulator of the apoptosome dysfunction induced by the impaired release of apoptogenic factors in the cytosol and/or the increased level of cytosolic antiapoptotic proteins. It may operate to amplify weak apoptotic signals induced by oxidative stress, ionizing radiation or drugs to sensitize tumor cells to chemotherapy. 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AFP positively regulates formation of Apaf-1 apoptosome in cell-free extracts and promotes caspase activation and release of caspase