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The Contribution of Apoptosis-inducing Factor, Caspase-activated DNase, and Inhibitor of Caspase-activated DNase to the Nuclear Phenotype and DNA Degradation during Apoptosis

2005; Elsevier BV; Volume: 280; Issue: 42 Linguagem: Inglês

10.1074/jbc.m504015200

ISSN

1083-351X

Autores

Vı́ctor J. Yuste, I. Prieto Sánchez, Carme Solé, Rana S. Moubarak, José R. Bayascas, Xavier Dolcet, Mario Encinas, Santos A. Susín, Joan X. Comella,

Tópico(s)

Global Peace and Security Dynamics

Resumo

We have assessed the contribution of apoptosis-inducing factor (AIF) and inhibitor of caspase-activated DNase (ICAD) to the nuclear morphology and DNA degradation pattern in staurosporine-induced apoptosis. Expression of D117E ICAD, a mutant that is resistant to caspase cleavage at residue 117, prevented low molecular weight (LMW) DNA fragmentation, stage II nuclear morphology, and detection of terminal deoxynucleotidyl transferase staining. However, high molecular weight (HMW) DNA fragmentation and stage I nuclear morphology remained unaffected. On the other hand, expression of either D224E or wild type ICAD had no effect on DNA fragmentation or nuclear morphology. In addition, both HMW and LMW DNA degradation required functional executor caspases. Interestingly, silencing of endogenous AIF abolished type I nuclear morphology without any effect on HMW or LMW DNA fragmentation. Together, these results demonstrate that AIF is responsible for stage I nuclear morphology and suggest that HMW DNA degradation is a caspase-activated DNase and AIF-independent process. We have assessed the contribution of apoptosis-inducing factor (AIF) and inhibitor of caspase-activated DNase (ICAD) to the nuclear morphology and DNA degradation pattern in staurosporine-induced apoptosis. Expression of D117E ICAD, a mutant that is resistant to caspase cleavage at residue 117, prevented low molecular weight (LMW) DNA fragmentation, stage II nuclear morphology, and detection of terminal deoxynucleotidyl transferase staining. However, high molecular weight (HMW) DNA fragmentation and stage I nuclear morphology remained unaffected. On the other hand, expression of either D224E or wild type ICAD had no effect on DNA fragmentation or nuclear morphology. In addition, both HMW and LMW DNA degradation required functional executor caspases. Interestingly, silencing of endogenous AIF abolished type I nuclear morphology without any effect on HMW or LMW DNA fragmentation. Together, these results demonstrate that AIF is responsible for stage I nuclear morphology and suggest that HMW DNA degradation is a caspase-activated DNase and AIF-independent process. DNA degradation is considered to be one of the defining hallmarks of apoptosis and is one of the first biochemical characteristics described for this type of cell death (1Wyllie A.H. Nature. 1980; 284: 555-556Crossref PubMed Scopus (4152) Google Scholar). DNA fragmentation is usually a two-step process in which the DNA is first cleaved into 50- to 300-kb fragments, termed high molecular weight (HMW) 8The abbreviations used are: HMWhigh molecular weightLMWlow molecular weightTUNELterminal deoxynucleotidyl transferase-mediated dUTP nick end labelingPBSphosphate-buffered salineAIFapoptosis-inducing factorCADcaspase-activated DNaseICADinhibitor of CADMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideSTPstaurosporinedsRNAdouble-stranded RNAZbenzyloxycarbonylFMKfluoromethyl ketoneCHEFclamped homogeneous electric fields electrophoresisQ-VD-OPhN-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone. DNA fragmentation. Subsequently, DNA is degraded into smaller fragments of oligonucleosomal size, known as low molecular weight (LMW) DNA degradation or DNA ladder (2Lecoeur H. Exp. Cell Res. 2002; 277: 1-14Crossref PubMed Scopus (177) Google Scholar). The early HMW degradation is associated with initial nuclear morphological changes characterized by a condensation of the chromatin around the nuclear membrane (stage I chromatin condensation or nuclear morphology). The later, LMW degradation coincides with a more advanced nuclear chromatin condensation into highly packed round masses (stage II chromatin condensation) (2Lecoeur H. Exp. Cell Res. 2002; 277: 1-14Crossref PubMed Scopus (177) Google Scholar). Although HMW DNA fragmentation usually precedes LMW DNA fragmentation, there are evidences indicating that apoptosis can proceed without LMW DNA degradation while still displaying the HMW (3Ucker D.S. Obermiller P.S. Eckhart W. Apgar J.R. Berger N.A. Meyers J. Mol. Cell Biol. 1992; 12: 3060-3069Crossref PubMed Scopus (234) Google Scholar, 4Oberhammer F. Wilson J.W. Dive C. Morris I.D. Hickman J.A. Wakeling A.E. Walker P.R. Sikorska M. EMBO J. 1993; 12: 3679-3684Crossref PubMed Scopus (1161) Google Scholar, 5Boix J. Llecha N. 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Chem. 2001; 276: 22323-22331Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). high molecular weight low molecular weight terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling phosphate-buffered saline apoptosis-inducing factor caspase-activated DNase inhibitor of CAD 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staurosporine double-stranded RNA benzyloxycarbonyl fluoromethyl ketone clamped homogeneous electric fields electrophoresis N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone. The execution of apoptotic cell death is governed by caspases, a family of cysteine proteases that, after activation by different pro-apoptotic stimuli, cleave target cell substrates (10Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2451) Google Scholar, 11Creagh E.M. Martin S.J. Biochem. Soc. Trans. 2001; 29: 696-702Crossref PubMed Scopus (0) Google Scholar). Caspases induce DNA degradation through the activation of the specific nuclease, caspase-activated DNase (CAD) (12Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2807) Google Scholar), also known as caspase-activated nuclease (13Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar) or DNA fragmentation factor 40 kDa (DFF40) (14Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (501) Google Scholar). CAD is sufficient to induce both stage II chromatin condensation and LMW DNA degradation in isolated nuclei (14Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (501) Google Scholar, 15Samejima K. Tone S. Kottke T.J. Enari M. Sakahira H. Cooke C.A. Durrieu F. Martins L.M. Nagata S. Kaufmann S.H. Earnshaw W.C. J. Cell Biol. 1998; 143: 225-239Crossref PubMed Scopus (110) Google Scholar). In growing cells, CAD remains inactive in the cytoplasm associated to the inhibitor of CAD (ICAD) (16Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1424) Google Scholar), also known as DNA fragmentation factor 45 kDa (DFF45) (17Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1649) Google Scholar). ICAD is encoded by alternatively spliced mRNAs that generate long (ICAD-L) and short (ICAD-S) forms of ICAD. ICAD has dual functions, acting both as a CAD inhibitor and as a chaperone for CAD synthesis. Therefore, the expression of CAD in the absence of co-expressed ICAD results in the generation of inactive aggregates of CAD (18Sakahira H. Enari M. Nagata S. J. Biol. Chem. 1999; 274: 15740-15744Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). ICAD harbors two caspase recognition sites at Asp117 and Asp224. CAD release from ICAD inhibition is achieved by cleavage of ICAD at these Asp residues by caspase-3 (14Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (501) Google Scholar, 17Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1649) Google Scholar, 18Sakahira H. Enari M. Nagata S. J. Biol. Chem. 1999; 274: 15740-15744Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 19Wolf B.B. Schuler M. Echeverri F. Green D.R. J. Biol. Chem. 1999; 274: 30651-30656Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). CAD is the best characterized nuclease implicated in apoptosis, but its specific contribution to apoptotic DNA processing and nuclear morphological changes has not been completely elucidated. Most studies on the role of ICAD in both HMW and LMW DNA fragmentation have been performed in ICAD knock-out mice or by expressing caspase-resistant forms of ICAD. Because ICAD acts as a chaperone during CAD synthesis, ICAD knock-out mice also lack a functional CAD. From these studies, the functional consequences of ICAD deficiency (20Zhang J. Liu X. Scherer D.C. van Kaer L. Wang X. Xu M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12480-12485Crossref PubMed Scopus (159) Google Scholar, 21Zhang J. Lee H. Lou D.W. Bovin G.P. Xu M. Biochem. Biophys. Res. Commun. 2000; 274: 225-229Crossref PubMed Scopus (25) Google Scholar) have been attributed to the lack of active CAD, and do not show HMW or LMW DNA fragmentation, a result comparable to that obtained with a CAD-/- mice (22Kawane K. Fukuyama H. Yoshida H. Nagase H. Ohsawa Y. Uchiyama Y. Okada K. Iida T. Nagata S. Nat. Immunol. 2003; 4: 138-144Crossref PubMed Scopus (194) Google Scholar). Additionally, human lymphoma cell lines overexpressing caspase-resistant ICAD also lack both LMW and HMW DNA fragmentation (23Sakahira H. Enari M. Ohsawa Y. Uchiyama Y. Nagata S. Curr. Biol. 1999; 9: 543-546Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Altogether, these results suggest that CAD is the enzyme also responsible for both HMW and LMW DNA fragmentation. However, it cannot be discarded that ICAD might have additional functions independent of CAD, and therefore, the phenotype of ICAD-/- cells could be different from that of those expressing caspase-resistant ICAD. In that sense, other reports have shown that CAD is dispensable for apoptotic HMW DNA fragmentation. Samejima et al. (15Samejima K. Tone S. Kottke T.J. Enari M. Sakahira H. Cooke C.A. Durrieu F. Martins L.M. Nagata S. Kaufmann S.H. Earnshaw W.C. J. Cell Biol. 1998; 143: 225-239Crossref PubMed Scopus (110) Google Scholar) have demonstrated that the activity causing nuclear morphological apoptotic changes could be inhibited by caspase inhibitors but not by ICAD. More significantly, CAD-deficient chicken DT40 cells failed to undergo LMW degradation and stage II chromatin condensation but displayed normal HMW DNA degradation and stage I morphology. These data suggest that factors other than CAD might be involved (24Samejima K. Tone S. Earnshaw W.C. J. Biol. Chem. 2001; 276: 45427-45432Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In summary, these results provide evidence that CAD triggers LMW DNA fragmentation, although the role of CAD and ICAD in HMW fragmentation remains controversial (25Nagata S. Nagase H. Kawane K. Mukae N. Fukuyama H. Cell Death Differ. 2003; 10: 108-116Crossref PubMed Scopus (360) Google Scholar). It has been suggested that endonucleases, other than CAD, could be responsible for HMW DNA fragmentation (24Samejima K. Tone S. Earnshaw W.C. J. Biol. Chem. 2001; 276: 45427-45432Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 28Parrish J.Z. Xue D. Mol. Cell. 2003; 11: 987-996Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 29Montague J.W. Hughes F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 6677-6684Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 30Nagata T. Kishi H. Liu Q.L. Yoshino T. Matsuda T. Jin Z.X. Murayama K. Tsukada K. Muraguchi A. J. Immunol. 2000; 165: 4281-4289Crossref PubMed Scopus (18) Google Scholar, 31Cande C. Vahsen N. Kouranti I. Schmitt E. Daugas E Spahr C. Luban J. Kroemer R.T. Giordanetto F. Garrido C. Penninger J.M. Kroemer G. Oncogene. 2004; 23: 1514-1521Crossref PubMed Scopus (228) Google Scholar, 32Lu Z.G. Zhang C.M. Zhai Z.H. Cell Res. 2004; 14: 134-140Crossref PubMed Scopus (4) Google Scholar, 33Sotolongo B. Huang T.T. Isenberger E. Ward W.S. J. Androl. 2005; 26: 272-280Crossref PubMed Scopus (105) Google Scholar). In the search for additional endonucleases that could be involved in DNA fragmentation, several molecules have been proposed. Among them, apoptosis-inducing factor (AIF) has been demonstrated to play a role in HMW DNA fragmentation (26Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3451) Google Scholar). AIF promotes caspase-independent HMW fragmentation and type I nuclear condensation when released from mitochondria by pro-apoptotic stimuli (27Susin S.A. Daugas E. Ravagnan L. Samejima K. Zamzami N. Loeffler M. Costantini P. Ferri K.F. Irinopoulou T. Prevost M.C. Brothers G. Mak T.W. Penninger J. Earnshaw W.C. Kroemer G. J. Exp. Med. 2000; 192: 571-580Crossref PubMed Scopus (661) Google Scholar). This protein lacks intrinsic nuclease activity, suggesting that AIF activates an unidentified nuclease responsible for DNA fragmentation (26Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3451) Google Scholar). Seven additional cell death-related nucleases (crn-1 to -6 and cyp-13) have been identified in Caenorhabditis elegans through functional genomic analysis, which, along with two known nucleases (CPS-6 and NUC-1), comprise at least two independent pathways that contribute to cell killing by degrading chromosomal DNA. Several of these proteins are components of important cellular processes such as RNA processing, protein folding, and DNA replication and repair, and appear to be important for the survival and proper development of the nematode (28Parrish J.Z. Xue D. Mol. Cell. 2003; 11: 987-996Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). One of the genes found, CYP-13, belongs to the family of cyclophilins, which have been previously involved in chromosomal DNA degradation in vertebrates. Cyclophilins show a nuclease activity distinct from the cis-trans-isomerase one, and their ionic requirements are similar to that described for apoptotic nucleases (29Montague J.W. Hughes F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 6677-6684Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 30Nagata T. Kishi H. Liu Q.L. Yoshino T. Matsuda T. Jin Z.X. Murayama K. Tsukada K. Muraguchi A. J. Immunol. 2000; 165: 4281-4289Crossref PubMed Scopus (18) Google Scholar). Cyp C has been shown to induce 50-kbp DNA fragmentation on isolated nuclei (29Montague J.W. Hughes F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 6677-6684Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), whereas Cyp A has been demonstrated to interact and cooperate with AIF in apoptosis-associated chromatinolysis (31Cande C. Vahsen N. Kouranti I. Schmitt E. Daugas E Spahr C. Luban J. Kroemer R.T. Giordanetto F. Garrido C. Penninger J.M. Kroemer G. Oncogene. 2004; 23: 1514-1521Crossref PubMed Scopus (228) Google Scholar). In addition, it has been shown that Xenopus egg extracts have a HMW DNA degradation activity activated by Caspase 3, which is different than the Xenopus CAD homolog, whose molecular identity remains uncharacterized (32Lu Z.G. Zhang C.M. Zhai Z.H. Cell Res. 2004; 14: 134-140Crossref PubMed Scopus (4) Google Scholar). The relationship of this activity with AIF has not been analyzed. Other poorly defined endonuclease activities capable of processing the DNA into 50-kb fragments have also been reported in spermatozoa (33Sotolongo B. Huang T.T. Isenberger E. Ward W.S. J. Androl. 2005; 26: 272-280Crossref PubMed Scopus (105) Google Scholar). We have previously demonstrated that IMR-5 neuroblastoma cells failed to display LMW DNA fragmentation due to a defect in the ICAD/CAD system that results in a non-functional CAD (9Yuste V.J. Bayascas J.R. Llecha N. Sanchez-Lopez I. Boix J. Comella J.X. J. Biol. Chem. 2001; 276: 22323-22331Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In the current report, we show that IMR-5 cells display HMW DNA fragmentation with undetectable TUNEL reactivity after staurosporine (STP) treatment. To ascertain the importance of ICAD in the nuclear apoptotic changes, we analyzed the contribution of the two caspase-target aspartic residues of ICAD (Asp117 or Asp224) in SH-SY5Y cells. We demonstrate that overexpression of the ICAD-D117E single mutant prevents LMW DNA fragmentation, stage II chromatin condensation, and detection of TUNEL staining, whereas HMW DNA degradation and stage I nuclear morphology remain unaltered. In cells overexpressing wild type ICAD or the ICAD-D224E single mutant no alterations to DNA fragmentation and nuclear morphology were observed. Overexpression of D117E-ICAD does not affect the release of AIF or cytochrome c from mitochondria to cytosol. We show for the first time that dsRNA knock-down of endogenous AIF expression in SH-SY5Y cells abolishes stage I nuclear condensation but not HMW or LMW DNA degradation. Similar results are obtained by intracellular delivery of blocking anti-AIF antibodies. We conclude that CAD is not responsible for HMW DNA degradation and stage I chromatin condensation, and this depends instead on the activity of other endonucleases that generate TUNEL-negative ends on DNA fragments. Our results, together with those reported using the ICAD-/- mice (21Zhang J. Lee H. Lou D.W. Bovin G.P. Xu M. Biochem. Biophys. Res. Commun. 2000; 274: 225-229Crossref PubMed Scopus (25) Google Scholar), further suggest a role for ICAD in HMW DNA degradation. Chemical Reagents—STP and Me2SO were purchased from Sigma. Z-VD(OMe)VAD(OMe)-FMK (caspase inhibitor-2), Z-D(OMe)E-(OMe)VD(OMe)-FMK (caspase inhibitor-3/7), Z-VE(OMe)ID(OMe)-FMK (caspase inhibitor-6), Z-IE(OMe)TD(OMe)-FMK (caspase inhibitor-8), Z-LE(OMe)HD(OMe)-FMK (caspase inhibitor-9), and Q-VD(non-OMe)-OPh (pan caspase inhibitor) were from Calbiochem (Barcelona, Spain). Plasmid Construction—The pCRII vector containing the full open reading frame of the long form of human ICAD (1.3 kb) was kindly provided by Dr. X. Wang (17Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1649) Google Scholar). Mutated forms of hICAD, hICAD-D117E and hICAD-D224E, were generated with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. The primers used for introducing the D117E mutation were, forward: 5′-ATT TCC CAA GAG TCC TTT GAC GTC GAT GAA ACA GAG AGC GGG GCA GGG TTG AAG-3′ and reverse 5′-CTT CAA CCC TGC CCC GCT CTC TGT TTC ATC GAC GTC AAA GGA CTC TTG GGA AAT-3′ (changed positions are indicated in bold). In these primers we also introduced a silent mutation that generates a unique restriction site for AatII (underlined). For the D224E mutation, primers were, forward 5′-G GAG GAG GTG GAT GCA GTA GAG ACG GAT ATC AGC AGA GAG ACC TCC-3′ and reverse GGA GGT CTC TCT GCT GAT ATC CGT CTC TAC TGC ATC CAC CTC CTC C) (changed positions are highlighted in bold). These primers also introduced a silent mutation that generates a restriction site for EcoRV (underlined). A FLAG epitope was introduced at 5′ position in the wild type and mutant ICAD by PCR using the primers HindIII-FLAG-hICAD (forward) 5′-CCC AAG CTT ATG GAC TAT AAG GAT GAC GAT GAC AAG GAG GTG ACC GGG GAC GCC GGG GTA-3′ and EcoRI-DFF45-R (reverse) 5′-CGG AAT TCT ATG TGG GAT CCT GTC TG GCT-3′. The PCR products were subcloned into the pcDNA3 expression vector (Invitrogen). All constructs were confirmed by DNA sequencing. Final constructs referred to as pcDNA3-hICAD-5′FLAG, pcDNA3-hICAD-D117E-5′FLAG, and pcDNA3-hICAD-D224E-5′FLAG. Empty vector (pcDNA3) was used as control. Cell Culture and Constitutive Transfection—Human SH-SY5Y and IMR-5 cell lines were cultured in Dulbecco's modified Eagle's medium (Invitrogen) with 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen). Cells were maintained at 37 °C in a saturating humidity atmosphere containing 95% air and 5% CO2. Cells were transfected with 3 μg of empty pcDNA3, pcDNA3-hICAD-5′FLAG, pcDNA3-hICAD-D117E-5′FLAG, or pcDNA3-hICAD-D224E-5′FLAG. Transfections were performed using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions. Stably transfected cells were obtained by selection with 500 μg/ml Geneticin (Invitrogen) and several clones, as well as the pool of transfected cells, were propagated and tested for ICAD expression and processing by Western blotting. Most of the experiments were carried out using pools of transfected cells. Clones gave comparable results. MTT Reduction and Trypan Blue Exclusion Cell Viability Assays and Chromatin Staining with Hoechst 33258—MTT is a water-soluble tetrazolium salt that is reduced by metabolically viable cells to a colored, water-insoluble formazan salt. The procedure employed for this assay was the same as that described by Boix et al. (5Boix J. Llecha N. Yuste V.J. Comella J.X. Neuropharmacology. 1997; 36: 811-821Crossref PubMed Scopus (77) Google Scholar). For trypan blue staining, cells were seeded in 24-multiwell plates at 5-3 × 104 cells/well. After 24 h of seeding, cells were treated with STP at the adequate doses and times. Then cells were gently dissociated with a blue tip in their own culture medium, and a sample (100 μl) was taken and mixed with 20 μl of trypan blue solution (0.4%) (Sigma). Ten microliters of the resulting cell suspension were counted with a hemocytometer. The results were expressed as percentages of trypan blue-stained cells over the total number of cells. Nuclear morphology was assessed by staining cells with the Hoechst 33258, which is also known as bisbenzimide (2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride) as established in our laboratory (5Boix J. Llecha N. Yuste V.J. Comella J.X. Neuropharmacology. 1997; 36: 811-821Crossref PubMed Scopus (77) Google Scholar). The normal or apoptotic cell nuclei were visualized with an Olympus microscope equipped with epifluorescence optics under UV illumination. dsRNA Preparation and Transfection—Two different dsRNA oligonucleotides against human AIF (R1, CCGGCUCCCAGGCAACUUGdTdT and R2, GCAUGCUUCUACGAUAUAAdTdT) and one scrambled sequence were bioinformatically designed and synthesized (Proligo France, Paris, France). Transfections of 5 × 105 cells with 10 μg of the indicated dsRNA were performed with Nucleofector (Amaxa, Cologne, Germany) using the G-04 program and Cell Line Nucleofector Solution V, following manufacturer's instructions. After 2 days, cells were replated in adequate culture dishes, depending on the experiment, and 72 h post transfection, the treatment and the corresponding protocol were performed. Protein Extractions and Western Blotting—When whole protein extracts were used, ∼1 × 106 cells per condition were detached from 35-mm culture dishes, gently pelleted by centrifugation and washed twice in phosphate-buffered saline (PBS). Cells were lysed in total extraction buffer containing 125 mm Tris-HCl, pH 6.8, and 2% SDS pre-warmed at 95 °C. Mitochondria-free cytosolic extraction of AIF and cytochrome c was performed with 2 × 106 cells seeded on 60-mm plates and treated with STP for the times indicated. After treatment, cells were detached, harvested in PBS, and resuspended in ten volumes of extraction buffer containing 220 mm mannitol, 70 mm sucrose, 50 mm Hepes-KOH (pH 7.2), 10 mm KCl, 5 mm EGTA, 2 mm MgCl2, and 1 mm phenylmethylsulfonyl fluoride, and kept on ice for 15 min. Cells were centrifuged in a Microfuge at 16,000 × g for 15 min at 4 °C, and the supernatant was retained. Nuclear and mitochondrial subfractionation extracts were performed with NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Perbio Science France, Brebières, France) following the manufacturer's instructions. In all cases, protein content was quantified by a modified Lowry assay (Bio-Rad Dc protein assay, Bio-Rad). Around 5-25 μg of protein per condition were electrophoresed in 15% (FLAG, ICAD, CAD, caspase-3, and cytochrome c detection), 10% (AIF detection), or 6% (α-fodrin and poly(ADP-ribose) polymerase detection) SDS-polyacrylamide gels that were electrotransferred to Immobilon-polyvinylidene difluoride membranes (Millipore, Bedford, MA) with a semidry apparatus (Hoefer, Amersham Biosciences). Filters were probed with the indicated primary antibodies and incubated with secondary antibodies conjugated with peroxidase (Sigma). As substrates for immunodetection we used either ECL (Amersham Biosciences) or SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology). Antibodies used in this study were anti-FLAG M2, anti-Hsp60, anti-actin, anti-cytochrome c, and anti-AIF (Sigma), anti-DFF45/ICAD (Stressgen Biotechnologies Corp., Victoria, BC, Canada), anti-α-spectrin/fodrin, anti-CAD (Chemicon International, Inc., Temecula, CA), anti-poly(ADP-ribose) polymerase C2.10 (Enzyme System Products, Livermore, CA), anti-caspase 3, anti-cleaved caspase-3, anti-panERK, anti-SOS-2 (BD Biosciences), and anti-fibrillarin (Abcam Ltd., Cambridge, UK). When required, the membranes were stained with Naftol Blue to assess comparable loading of lanes. TUNEL Assay and Nuclei Staining—SH-SY5Y and IMR-5 cells were treated with staurosporine as indicated in figure legends. Detection of blunt double-stranded fragments carrying a 5′-phosphate and 3′-hydroxyl group was carried out by fixing cells in freshly prepared 1% paraformaldehyde for 30 min at 4 °C and permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate for 10 min at 4 °C. After washing in PBS containing 0.1% Triton X-100, they were incubated with 50 μl of a reaction mixture containing 0.025 nmol of Fluorescein-12-dUTP, 0.25 nmol of dATP, 2.5 mm CoCl2, 40 units of recombinant terminal deoxynucleotidyl transferase (TdT), and TdT reaction buffer from Roche Applied Science for 1 h at 37°C. The reaction was stopped by adding 20 mm EGTA. Cells were washed twice with PBS, stained with 0.05 μg/ml Hoechst 33258, mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA), and photographed under fluorescein isothiocyanate and UV illumination in an inverted epifluorescence microscope (Olympus IX70-S8F2) coupled to a camera (model OM-4 Ti; Olympus). Electron Microscopy—Approximately 3 × 106 cells per condition were detached from 60-mm culture dish, gently pelleted by centrifugation, and washed with PBS. Pellets were fixed at 4 °C with 2.5% glutaraldehyde in 0.1 m phosphate buffer, pH 7.4, rinsed three times with 0.1 m phosphate buffer, post-fixed in 1% osmium tetroxide (OsO4), dehydrated in graded acetone, and embedded in Durcupan ACM Epoxy resin (Fluka, Buchs, Switzerland). Ultrathin sections of selected areas were obtained, mounted in copper grids, counterstained with uranyl acetate and lead citrate, and observed with a Zeiss EM910 electron microscopy (Carl Zeiss Microscope Systems, Oberkochen, Germany). DNA Degradation Analysis—High molecular DNA fragmentation was assayed by clamped homogeneous electric fields (CHEF) electrophoresis using a CHEF-DR (Bio-Rad) apparatus. Approximately 2 × 106 cells per condition were suspended in 40 μl of PBS, warmed at 60 °C for 5 min, mixed with an equal volume of warm 1% low melting point agarose in 0.5 × TBE buffer (45 mm Tris, 45 mm boric acid, 1.0 mm EDTA, pH 8.3), transferred to cold agarose block formers and left at 4 °C for 30 min. Agarose blocks were removed from the block formers and incubated at 50 °C firstly for 24 h in 1 ml of NDS buffer (1% lauryl-Sarkosyl, 10 mm Tris, 0.5 m EDTA, pH 9.5) containing 200 μg/ml proteinase K with gentle shaking. The NDS supernatants were further processed for internucleosomal DNA degradation (LMW DNA degradation). Next, the blocks were further incubated for 24 h with the same buffer containing 10 μg/ml RNase A. After two 1-h washes at room temperature in 0.5 × TBE buffer, the blocks were inserted into wells of a 1% CHEF agarose gel in 0.5 × TBE. Electrophoresis was carried out at 6 V/cm for 14 h at 14 °C with a switch time of 5-50 s. For LMW DNA degradation, equal volumes of NDS supernatants and cold 100% ethanol were mixed, and the mixture was incubated at -20 °C overnight and centrifuged at maximum speed for 10 min at 4 °C. Pellets were washed once in 70% ethanol and resuspended in TE buffer (10 mm Tris-HCl, pH 9.0, 1 mm EDTA) containing 200 μg/ml RNase 15 min at 60 °C. DNA was analyzed in 1.5% agarose gel in TAE buffer (1 mm EDTA, 40 mm Tris acetate, pH 7.6) stained with ethidium bromide. Cell cycle analysis and sub-G1 peak of DNA fragmentation were performed using propidium iodide staining. Briefly, after treatments, cells were recovered, rinsed with PBS, and resuspended with PBS containing 0.5 μg/ml propidium iodide, 0.1% Igepal CA-630, and 50 μg/ml DNase-free RNase. After 20 min of incubation, DNA content was determined using a FACSCalibur (BD Biosciences, Oxford, UK) flow cytometer. Data were analyzed by CellQuest software. Intracellular Antibody Delivery Approa

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