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1 Institut National de la Santé et de la Recherché Médicale U-517, Faculty of Medicine and Pharmacy, Dijon, France;
2 Centre National de la Recherché Scientifique UMR 1599, Institute Gustave Roussy, Villejuif, France;
3 Department of Chemistry, Queen Mary-University of London, London, United Kingdom;
4 Molecular Modeling and Design, Pharmacia, Nerviano, Italy;
5 Apoptosis Laboratory, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark;
6 Ontario Cancer Institute, Department of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario, Canada;
7 Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri;
8 Reproductive Toxicology Division (MD-72), National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina; and
9 Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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AIF is synthesized as a Mr 67,000 precursor and converted to mature AIF (Mr 57,000) upon mitochondrial import and removal of the NH2-terminal mitochondrial localization signal (8) . Mature AIF is a flavoprotein with NADH reductase activity and a characteristic glutathione reductase-like crystal structure (14 , 15) . The flavin adenine dinucleotide cofactor is dispensable for the apoptogenic function but required for the oxidoreductase activity of AIF (15 , 16) , suggesting that AIF is a bifunctional protein with a mitochondrial resident role and an independent apoptogenic function. Upon release from mitochondria, AIF is actively imported into the nucleus (8) , where it interacts with chromatin (17) , provoking its condensation and large scale DNA fragmentation in a caspase-independent fashion (18 , 19) . AIF possesses clusters of positive surface charges allowing it to interact with DNA and to induce chromatin condensation yet are dispensable for its oxidoreductase activity (17) . Although it has been suggested that AIF may have also an antioxidant (and hence antiapoptotic) function (20) , the knockdown of the AIF gene in Caenorhabditis elegans has confirmed the preponderant proapoptotic function of AIF (21) . Recombinant C. elegans AIF can cooperate with an endonuclease to degrade DNA in vitro (21) .
Stress-inducible HSP70 is a prominent cytoprotective factor. Under normal conditions, HSP70 functions as an ATP-dependent chaperone by assisting the folding of newly synthesized proteins and polypeptides, the assembly of multiprotein complexes, and the transport of proteins across cellular membranes (22, 23, 24) . HSP70 up-regulation, as a consequence of either cellular stress or transfection, inhibits apoptosis induced by a wide range of insults and may contribute to oncogenic transformation (25 , 26) . Thus, HSP70 overexpression increases the tumorigenicity of cancer cells in rodent models (27) and correlates with poor prognosis in breast cancer (28) . Conversely, HSP70 down-regulation is sufficient to kill tumor cells or to facilitate the induction of apoptosis in vitro (29) and reduced tumorigenicity in vivo (30) . The antiapoptotic function of HSP70 involves interactions with several components of the apoptotic machinery. HSP70 has been demonstrated to bind to Apaf-1, thereby preventing the recruitment of procaspase-9 to the apoptosome (31) . We have shown that HSP70 can also inhibit apoptosis by directly interacting with and neutralizing AIF (32) .
In this work, we constructed deletion mutants of AIF and mapped the AIF domains involved in HSP70 binding. We demonstrated that a region comprised between amino acids 150 and 228 of AIF was necessary for HSP70 binding. Computer calculations of the interaction between HSP70 and AIF indeed favored this conclusion. On the basis of this information, we constructed an AIF-derived construct that maintained the region required for HSP70 binding was exclusively cytosolic and noncytotoxic. We demonstrated that this AIF-derived decoy for HSP70 had chemosensitizing properties based on its capacity to interact with and to neutralize endogenous HSP70.
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MATERIALS AND METHODS |
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Construction of Vectors.
The AIF-GFP pcDNA3 construct has been described previously (16)
. The deletions described in this article were performed using the QuikChange Site-directed and the Exemple-site Mutagenesis kits (Stratagene, La Jolla, CA) following the manufacturers recommendations. The resulting plasmids were verified by enzymatic digestion. Mutants were also cloned in a pcDNA3 vector carrying a His, V5-epitope tag (Invitrogen, Carlsbad, CA).
Molecular Modeling.
On the basis of the structures of mouse AIF [pdb code: 1GV4 (14)
] and HSC70 [pdb code: 7hsc, (35)
], the corresponding homology models for human AIF and HSP70 was generated. Molecular surfaces and curvatures were calculated using Grasp (36)
. Guided by surface complementarity in terms of curvature, as well as electrostatic and hydrophobic properties (37)
, initial models of the human AIF-HSP70 complex were generated. These complexes were refined by energy minimization and were scored by interaction energies and visual inspection of the interactions. The best scoring complex was chosen for additional analysis.
Transient Transfection and Cytotoxicity Assays.
Cells (5–7 x 103/well) were seeded on to 96-well plates 1 day before transfection. Transient transfections with AIF-Wt, mutants, or GFP alone were carried out using Superfect reagent (Qiagen, Courtaboeuf, France) according to manufacturers protocol. Cells were treated or not with the specified drugs 24 h after transfection. The number of viable cells after treatment was measured by a crystal violet colorimetric assay after specified duration of treatment.
Cytometric Analysis.
Transiently transfected cells were treated or not with staurosporine for 24 h. Cell death was measured in transfected cells after propidium iodide staining of nonpermeabilized cells. Ten thousand cells were analyzed using FACS Scan flow cytometer (Becton Dickinson, Franklin Lakes, NJ), and the percentage of cell death was measured in GFP-labeled cells using two color flow cytometry.
Immunohistochemistry and Cell Morphological Studies.
A Leitz microscope equipped with an epi-illuminator and appropriate filters (Leica, Bron, France) was used for immunofluorescent and morphological studies. Subcellular localization of wild-type AIF-GFP and mutants and chromatin condensation with or without an apoptotic stimulus was performed after transient transfection. A total of 4 x 105 was seeded onto Lab-Tek 8-chamber slides a day before transfection. Cells were treated or not with staurosporine 24 h after transfection. GFP subcellular localization and chromatin condensation were determined as follows. Cells were washed once with PBS, fixed with 3% paraformaldehyde, and stained with Hoechst 33342 (1 mg/ml) for 30 min at 37°C. A total of 300 GFP-containing cells were counted to determine the Wt-AIF and mutant AIF subcellular localization and apoptosis.
Immunofluorescent labeling of endogenous AIF was performed in whole cells fixed in 3% paraformaldehyde and permeabilized in saponin (0.1% v/v in PBS-BSA). Appropriate concentration of AIF antibody (Sigma-Aldrich) was added to the cells in 100 µl of PBS-BSA. This antibody that recognizes the COOH-terminal 593-612 AIF sequence did not recognize ADD70 or ADD-Co. After 1 h of incubation at room temperature and two washes, cells were incubated with 568-Alexa (red) goat antimouse antibody (Interchim; Molecular Probes). Slides were washed three times with PBS and covered with coverslips.
Immunoblot Analysis.
Whole cell lysates were prepared by lysing the cells in 2% SDS, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and NaCl/Pi (pH 7.4) at 68°C for 5 min. Protein concentration was measured by the micro BCA protein assay (Pierce, Asnières, France). Proteins were separated in 8–12% SDS-polyacrylamide gel and electroblotted onto polyvinylidene difluoride membranes (Bio-Rad, Ivry sur Seine, France). Immunoblot analysis was performed using specific antibodies and enhanced chemoluminescence-based detection (Amersham, Buckinghamshire, United Kingdom, and Les Ullis, France). The antibodies used were the mouse monoclonal antihuman HSP70 and polyclonal antihuman HSP70, (StressGen, Victoria, British Columbia, Canada) and rabbit polyclonal antibodies raised against the COOH-terminal of mature AIF (amino acids 593–612; Sigma-Aldrich). As a result, this polyclonal antibody cannot recognize ADD70 or ADD-Co.
Cell Fractionation and Immunoprecipitation in Cell-Free Extracts.
Protein-protein interactions between the Wt-AIF or the AIF mutants and HSP70 were determined by immunoprecipitation experiments in nuclei-free, mitochondria-free cytosolic extracts from MEF-70 cells in which the in vitro translated tagged AIF protein has been added. In vitro translation of the His-V5 or GFP-tagged Wt-AIF and mutant AIF was performed using the TNT-coupled reticulocyte lysate systems (Promega, Charbonnières, France). ß-Galactoside tagged with the His-V5 epitope was used as a negative control for the immunoprecipitation reactions. The nuclei-free, mitochondria-free extracts were prepared as described previously (38)
. Briefly, cells were washed in ice-cold PBS (pH 7.2), then in hypotonic extraction buffer [HEB: 50 mM PIPES (pH 7.4), 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, and 0.1 mM phenylmethylsulfonyl fluoride] and centrifuged. The pellet was resuspended in HEB and lysed in a Dounce homogenizer. This cell lysate was centrifuged 30 min at 16,000 x g at 4°C, and the clarified supernatant was either tested immediately or stored in aliquots at -80°C. For immunoprecipitation, 50 µl of the cell-free extract was incubated with 20 µl of the reticulocyte lysate for each of the immunoprecipitation (IP) reactions at room temperature for 30 min. Immunoprecipitations were subsequently performed in a final total volume of 500 µl of immunoprecipitation buffer [50 mM HEPES (pH 7.6), 150 mM NaCl, 5 mM EDTA, and 0.1% NP40] with a polyclonal antihuman HSP70 antibody (1:100) with constant agitation at 4°C. The immunocomplexes were precipitated with protein A-Sepharose (Amersham). The pellet was washed five times and prepared for immunoblotting. Cytosolic and nuclear fractions for AIF studies were prepared by resuspending cells in ice-cold buffer A [250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 17 µg/ml phenylmethylsulfonyl fluoride, 8 µg/ml aprotinin, and 2 µg/ml leupeptine (pH 7.4)]. Homogenization was performed in a Potter-Thomas homogenizer. Nuclei were pelleted via a 10-min, 750 x g spin. The supernatant was spun at 10,000 x g for 25 min and the mitochondrial fraction resuspended in buffer A.
Measurement of Caspase-3 and Caspase-9 Activity.
Exponentialy growing cells were treated with cisplatin (Roger Bellon). At different times, caspase-3 and caspase-9 activity was assessed by the cleavage of the fluorometric substrates DEVD-AFC and LEHD-AFC, respectively (Calbiochem, San Diego, CA), from cell lysates in the presence or absence of nonspecific caspase inhibitory peptide ZVAD-fmk. AFC released from the substrates were excited at 400 nm. Emission was measured at 505 nm.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. Among the panel of deletion mutants of AIF, only those lacking amino acids 150–262 (AIF
150-268 and AIF122-262), were found to lose the HSP70 binding capacity (Fig. 1, B and E)
. Because AIF228-237 and AIF228-347 did bind to HSP70 in immunoprecipitation assays (Fig. 1B)
, we conclude that the AIF domain necessary for HSP70 binding is comprised between amino acids 150 and 228. Computer calculations of the interaction between HSP70 and AIF indeed favored a model of dense hydrophobic and electrostatic interactions (Fig. 1, C and D)
in which the segment of human AIF that participates in the interface comprises residues D184–E221, whereas the substrate-binding domain of HSP70 would participate with amino acids D534–K541, Y433–N436, and A469–V473 (Fig. 1D)
. This is in accordance with our previous results that demonstrated that the substrate binding domain of HSP70 but not the ATP-binding domain was necessary for HSP70 interaction with AIF (32)
. Human AIF K194 (which corresponds to mouse R193) is engaged in a salt bridge with HSP70 E532 (Fig. 1D)
. In contrast, the adjacent R192 of human AIF (which is not conserved in mouse AIF) is not involved in a specific interaction with HSP70, although it contributes to the packing in the interface. Driven by this computer simulation, we show that in vitro translated hAIF R192AK194A, a gain-of-function point mutant of AIF [mutation 192 (17)
], fails to interact with HSP70 in conditions in which wild-type hAIF does interact with HSP70 (Fig. 1E)
. Taken together, these findings underscore that a domain of AIF comprised between amino acids 150 and 228 engages in a tight molecular interaction with the substrate-binding domain of HSP70.
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1-100), the putative nuclear localization signal (NLS, 367-466), as well as the COOH-terminal region (567-609) previously described to be necessary for AIF apoptotic features (Ref. 16
; Fig. 2A
). As a control, we used a similar deletion mutant from which we further removed the region necessary for HSP70 binding (ADD-Co, Fig. 2A
). The capacity of ADD70 to bind HSP70, as opposed to ADD-Co, was controlled by coimmunoprecipitation (Fig. 2B)
. Microscopic analyses of MEF cells transfected with ADD70 demonstrate the exclusive cytosolic localization of ADD70 even after an apoptotic stimulus that induces the nuclear localization of AIF (Fig. 2C)
. We then examined if ADD70 was bound to HSP70 under these circumstances. Cells transfected with ADD70 and ADD-Co were immunoprecipitated using anti-V5, which recognizes the V5 tag introduced into ADD70 and ADD-Co. The unbound HSP70 level was subsequently determined by Western blotting of the supernatants. Results shown in Fig. 2D
indicate that HSP70 binds to ADD70 in cells, and as a consequence, the unbound level of HSP70 is less important in the ADD70-transfected cells than in the ADD-Co-transfected cells. This effect of ADD70 on HSP70 does not seem to provoke a compensatory induction of HSP70 or other inducible antiapoptotic family members because the basal expression of HSP70 (inducible and cognate), HSP27, HSP90, Bcl-2, and BclXL is similar in ADD70-, ADD-Co-, and mock-transfected MEF cells, as determined by Western blot (data not shown).
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. However, ADD70 but not ADD-Co sensitized MEF to STS-induced apoptosis (Fig. 3A)
. To determine the relative contribution of HSP70 to ADD70-mediated cell killing, we evaluated the capacity of ADD70 to kill MEF overexpressing HSP70 (MEF-70) and MEF originating from mice deficient for the two genes that code for inducible HSP70 (HSP70.1, HSP70.3). As already demonstrated (32)
, the apoptogenic activity of AIF-Wt correlated with the level of HSP70, meaning that it was more pronounced in those cells that lack HSP70 and less pronounced in cells overexpressing HSP70. Concerning ADD70, the expression level of HSP70 had profound effects on its sensitization potential. ADD70 lost its capacity to sensitizes to STS-induced cell death in MEF lacking HSP70 expression (Fig. 3B)
. In HSP70 overexpressing cells, the effect of ADD70 was slightly attenuated (Fig. 3C)
, which may reflect the fact that the quantity of antiapoptotic HSP70 that needs to be neutralized is higher than in wild-type MEF cells (32)
. Importantly, the ADD70-mediated sensitization to apoptosis induction was observed with a large panel of apoptosis inducers, including DNA damaging agents (cisplatin, VP16), serum depletion, vinblastin, and menadione, and no such effect was observed with ADD-Co (Fig. 3D)
. In conclusion, ADD70 can sensitize to cell death induction by virtue of its capacity to interact with HSP70.
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. Simultaneously, cytosolic extracts from ADD70-transfected cells stimulated with apoptosis inducers (STS or cisplatin) exhibited a much higher capacity to digest caspase-3 and caspase-9 substrates (Ac-DEVD-AFC and Ac-LEHD-AFC, respectively) as compared with cells transfected with ADD-Co (Fig. 4B)
. Moreover, by using an anti-AIF raised against an epitope do not present in ADD70 or ADD-Co, we demonstrated that MEF transfected with ADD70, after 12 h of treatment with STS, exhibited an enhanced nuclear translocation of endogenous AIF, as compared with ADD-Co-transfected cells (Fig. 5A)
. ADD70 inhibits the interaction of HSP70 with AIF (Fig. 5B)
most probably as a consequence of its capacity of occupying the substrate binding domain of HSP70. Hence, ADD70 acts at several distinct levels to favor apoptosis induction, namely at the level of AIF and at the level of caspase activation. We then tested whether ADD70 sensitized different human cancer cell lines (leukemic U937, colon cancer HT-29 and SW480, breast cancer MCF7, and cervix cancer HeLa cells) to STS and cisplatin treatment. We found the chemosensitizing potential of ADD70 extended to all of the cancer cell lines tested (Fig. 6)
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defined as necessary for HSP70 binding. Experiments performed with this construct indicate that this new molecule behaves like ADD70: (a) it binds to HSP70 (Fig. 7B)
; (b) lacks any apoptogenic function; and (c) increases the sensitivity of MEF and different cancer cells to chemotherapeutic agents (Fig. 7C)
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As shown here, we demonstrate that a rationally engineered decoy target of HSP70, ADD70, can sensitize cancer cells to apoptosis induction. ADD70 has no intrinsic apoptotic-stimulatory activity but exerts its chemosensitizing effect through the neutralization of HSP70, as indicated by the fact that its sensitization potential is abolished by deleting its HSP70 binding domain (aa 150–228). Importantly, ADD70 loses its apoptosis sensitizing effect in cells lacking HSP70, thus providing compelling evidence that ADD70 predisposes to apoptosis induction through its capacity to neutralize HSP70.
The fact that an AIF mutant lacking its intrinsic apoptogenic function can sensitize cells to apoptosis induction, including caspase-dependent apoptosis, has interesting theoretical implications. AIF has the capacity to induce some features of apoptosis in a caspase-independent fashion. In particular, it appears that AIF, once it translocates to the nucleus, can directly interact with DNA, thereby favoring chromatin condensation in reaction that does not involve caspases (16, 17, 18) . In addition, as shown here, AIF can bind and neutralize HSP70 in a fashion that favors caspase activation indirectly, presumably by blocking the inhibitory HSP70-Apaf-1 interaction (31) . This observation suggests that the caspase-dependent and caspase-independent pathways of apoptosis engage in a mutual cross-talk at several levels. Beyond the common Bcl-2 family protein-mediated control of the mitochondrial release of both caspase activators (cytochrome c and Smac/DIABLO) and caspase-independent death effectors (AIF, HtrA2, and endonuclease-G; Refs. 42, 43, 44 ), AIF may contribute to caspase activation at the postmitochondrial level by neutralizing the caspase-inhibitory function of HSP70.
Irrespective of these theoretical considerations, it appears that a positive strategy aimed at interfering with HSP70, as opposed to negative strategies based on antisense constructs or perhaps RNA interference, is feasible for chemosensitization, at least in vitro. Whether this approach is also feasible in vivo, for instance, by using ADD70-expressing adenovirus constructs awaits additional confirmation.
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FOOTNOTES |
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Grant support: Institut National pour la Santé et la Recherché Médicale (INSERM), the Comité Départemental de la Nièvre de la Ligue National contre le Cancer, the Association pour la Recherché contre le Cancer (ARC) Grants 9567 and 5204, the Conseil Régional de Bourgogne (to C. G.), Ligue Nationale contre le Cancer and the European Commission Grant QLG1-CT-1999-00739 (to G. K.). E. S. is supported by the CHU Le Bocage of Dijon, A. P. by a fellowship from the Ministère de l’Education Nationale, S. G. by a Poste vert from the INSERM, and C. C. by a fellowship from ARC.
Present address: C. G. and G. K. share senior coauthorship.
Requests for reprints: Carmen Garrido, INSERM U517, Faculty of Medicine and Pharmacy, 7 boulevard Jeanne d’Arc, 21033 Dijon, France. Phone: 33-3-80-39-32-84; Fax: 33-3-80-39-34-34; E-mail: cgarrido{at}u-bourgogne.fr
10 The abbreviations used are: Apaf-1, apoptotic protease activation factor-1; MEF, mouse embryonic fibroblast; AIF, apoptosis-inducing factor; HSP, heat shock protein; FBS, fetal bovine serum; GFP, green fluorescent protein; MS/MS, tandem mass spectrometry; AIF-Wt, AIF wild-type; STS, staurosporine. 
Received 3/13/03. Revised 7/17/03. Accepted 9/23/03.
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), transfected with ADD-Co (
), or with ADD70 (
), were treated for 24 h with STS (100 nM), cisplatin (CDDP, 5 µg/ml), etoposide (VP16, 100 µM), vinblastin (Vb, 10 ng/ml), menadione (Md, 25 µM), or cultured in the absence of serum (SD). Cell death was measured by cytofluorometry after staining of the cells with vital dye propidium iodide and while gating on the GFP+ population. Bars, SDs (n = 3).
