the role of apaf-1, caspase-9, and bid proteins in etoposide- or ... · apaf-1 or caspase-9 has...

[CANCER RESEARCH 60, 1645–1653, March 15, 2000]

The Role of Apaf-1, Caspase-9, and Bid Proteins in Etoposide- or Paclitaxel-inducedMitochondrial Events during Apoptosis

Charles L. Perkins, Guofu Fang, Caryn Nae Kim, and Kapil N. Bhalla1

Division of Clinical and Translational Research, Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, Florida 33136

ABSTRACT

Ectopic overexpression of Apaf-1 (2.5-fold) in human acute myeloge-nous leukemia HL-60 cells (HL-60/Apaf-1 cells) induced apoptosis andsensitized HL-60/Apaf-1 cells to etoposide- and paclitaxel-induced apo-ptosis (C. Perkinset al., Cancer Res.,58: 4561–4566, 1998). In this report,we demonstrate that in HL-60/Apaf-1 cells, the activity of caspase-9 and -3induced by Apaf-1 overexpression was associated with a significant in-crease (5-fold) in the cytosolic accumulation of cytochromec (cyt c), lossof mitochondrial membrane potential (DCm), and an increase in thereactive oxygen species. These were also associated with the processing ofprocaspase-8 and Bid (cytosolic, proapoptotic BH3 domain containingprotein). Transient transfection of Apaf-1 into the Apaf-1-containingmouse embryogenic fibroblasts (MEFs; Apaf-11/2MEFs) or Apaf-12/2MEFs also induced the processing of procaspase-9 and procaspase-8, Bidcleavage, and apoptosis. These events were secondary to the activity of thedownstream caspases induced by Apaf-1. This conclusion is supported bythe observation that in HL-60/Apaf-1 cells, ectopic expression of dominantnegative caspase-9, its inhibitory short isoform caspase-9b, or XIAP ortreatment with the caspase inhibitor zVAD (50 mM) inhibited Apaf-1-induced caspase-8 and Bid cleavage, mitochondrialDCm, release of cytc,and apoptosis. In contrast, a transient transfection of dominant negativecaspase-8 or CrmA or exposure to caspase-8 inhibitor zIETD-fmk inhib-ited the processing of procaspase-8 and Bid but did not inhibit thecytosolic accumulation of cytc in either the untreated HL-60/Apaf-1 cellsor the etoposide-treated HL-60/Apaf-1 and HL-60/neo cells. These resultsindicate that Apaf-1 overexpression lowers the apoptotic threshold byactivating caspase-9 and caspase-3. This triggers the mitochondrialDCmand cyt c release into the cytosol through a predominant mechanism otherthan cleavage of caspase-8 and/or Bid. This mechanism may involve acytosolic mitochondrial permeability transition factor, which may be pro-cessed and activated by the downstream effector caspases, thereby com-pleting an amplifying feedback loop, which triggers the mitochondrialevents during apoptosis.

INTRODUCTION

The family of mammalian caspases (aspartate-specific cysteineproteases) represents the effector arm of the apoptotic program (1, 2).Intracellularly, caspases exist as inactive zymogens (procaspases) thathave NH2-terminal prodomains plus large and small catalyticallyactive subunits. Caspases may be subclassified as initiators (e.g.,caspase-8, -10, -2, or -9) or effectors, also known as executioners(e.g.,caspase-3, -6, or -7), based on whether they have a large or smallprodomain (1, 2). Caspases with large prodomains interact with sig-naling adaptor molecules through motifs in the prodomains calledCARDs2 (1, 2). For example, interaction between CARDs in the

adaptor molecule FADD and the prodomain of procaspase-8 allowsthe formation of a death-inducing signaling complex during the Fasreceptor-induced signaling for apoptosis (3). Recruitment and oli-gomerization, followed by processing and activation of caspase-8 inthe Fas receptor-initiated death-inducing signaling complex, ulti-mately result in the cleavage and translocation of the cytosolic,proapoptotic BH3 domain-containing Bid protein to the mitochondria(4–7). Here it triggers the preapoptotic mitochondrial events leadingto the activation of the executioner, caspase-3 (4, 5). Previousin vitrostudies have demonstrated that the enforced expression of adaptormolecules FADD or CRADD triggers the cleavage and activation ofexecutioner caspase-3 and apoptosis by causing oligomerization of theinitiator caspases, caspase-8 and caspase-10, or caspase-2, respec-tively (8, 9). The generation of active caspases requires cleavage at theinternal Asp residue present between the catalytic large and smallsubunit, with further processing at the Asp residue present in theinterdomain linkers between the prodomain and the large subunit toremove the prodomain (1, 2). Once activated, caspases can cleavetheir substrates and other procaspases to generate active subunits (1,2). The executioner caspases can also proteolytically cleave a numberof cellular proteins,e.g.,PARP, lamins, DFF, fodrin, gelsolin, proteinkinase Ca, Rb, DNA-PK, and so forth, resulting in the morphologicalfeatures and DNA fragmentation of apoptosis (1, 2, 10).

Recently, Apaf-1 was identified as the human homologue of theCaenorhabditis elegansCED-4 protein and was shown to participateas an adaptor molecule in the sequential activation of caspase-9 andcaspase-3 (11–13). The NH2-terminal region of Apaf-1 contains aCARD, which can bind to the corresponding motif in procaspase-9.Apaf-1 also contains a central region homologous to the proapoptoticCED-4 protein, including a conserved P-loop, and a COOH-terminallong WD repeat domain that lacks homology with CED-4. In thepresence of dATP and cytc released from the mitochondria by anumber of apoptotic stimuli, the monomeric Apaf-1 is transformedinto an oligomeric complex made of at least eight subunits (14–16).This Apaf-1-cytc complex can bind and process procaspase-9, fol-lowed by the release of mature caspase-9 (15, 17). This, is turn, canprocess procaspase-3, triggering the caspase cascade of activities thatresults in apoptosis (15, 17). Recent studies have demonstrated that anendogenous alternately spliced isoform of caspase-9 (caspase-9b), amutation in the active site of caspase-9, or the deletion of caspase-9from the S-100 fraction of cytosol blocks cyt-c-induced and Apaf-1-mediated ultimate cleavage and activation of caspase-3 (12, 13, 18,19). The importance of Apaf-1-induced activity of caspase-9 followedby caspase-3 has been highlighted by reports that demonstrate thatApaf-1 or caspase-9 deficiency results in embryonic lethality due todefective neuronal apoptosis (20–23). Furthermore, inactivation ofApaf-1 or caspase-9 has been shown to substitute for p53 loss inpromoting oncogenic transformation of the Myc-expressing cells (24).

Similar to the enforced overexpression of FADD and CRADD,Apaf-1 overexpression was shown to induce apoptosis, through thecleavage and activation of caspase-9 and caspase-3 (25). Higher levelsof Apaf-1 were demonstrated to enhance apoptosis induced by che-motherapeutic agents,e.g., etoposide and Taxol, by increasing thesequential cleavage and activities of caspase-9 and caspase-3 (25).However, in these studies, the role of mitochondrialDCm and cytc

Received 11/12/99; accepted 1/19/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Division of Clinical andTranslational Research, Sylvester Comprehensive Cancer Center, University of MiamiSchool of Medicine, 1550 NW 10th Avenue (M710), Miami, FL 33136. Phone: (305)243-5907; Fax: (305) 243-5885; E-mail: [email protected].

2 The abbreviations used are: CARD, caspase recruitment domain; cytc, cytochromec; ROS, reactive oxygen species; MEF, mouse embryonic fibroblast; DN, dominantnegative; MPTF, mitochondrial permeability transition factor; PARP, poly(ADP-ribose)polymerase; DFF, DNA fragmentation factor; TRAIL, tumor necrosis factor-relatedapoptosis-inducing ligand; TNF, tumor necrosis factor; MMP, mitochondrial membranepotential; PI, propidium iodide.

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release in Apaf-1-mediated sensitization of apoptosis was not deter-mined. In the present studies, we investigated whether the lowering ofthe threshold to trigger apoptosis through Apaf-1 overexpressioninvolves procaspse-8 and Bid cleavage and activity, resulting in themitochondrialDCm and the release and cytosolic accumulation of cytc. Our findings demonstrate that Apaf-1 overexpression mediatesmitochondrialDCm, cytosolic accumulation of cytc, and apoptosispredominantly through a mechanism that involves the activity ofcaspase-9 and caspase-3 but not caspase-8 and Bid.

MATERIALS AND METHODS

Reagents.z-VAD-fmk, z-IETD-fmk, and z-LEHD-fmk were purchasedfrom Enzyme Systems Products (Livermore, CA). Anti-Apaf-1 and Anti-Bidantisera (5, 11) as well as cDNA of Apaf-1 and CrmA were kindly providedby Dr. Xiadong Wang (University of Texas, Southwestern School of Medicine,Dallas, TX). The recombinant human homotrimeric TRAIL (leucine zipperconstruct) was a gift from Immunex Corp. (Seattle, WA). Dr. Emad Alnemri(Thomas Jefferson University, Philadelphia, PA) kindly provided us with thecDNA of caspase 9b, DN caspase-9, and Apaf-1L, which possesses an addi-tional WD repeat.

Cells and Transfection of the Genes.Human myeloid leukemia HL-60cells were cultured as described previously (26). Viable HL-60 cells (2–3 3 106) were transiently transfected with 0.5–1.0mg each of the indicatedplasmid DNA using LipofectAMINE PLUS reagent (Life Technologies, Inc.,Gaithersburg, MD). Stable HL-60 cell lines overexpressing the human Apaf-1protein were developed as described previously (25). Lysates from the selectedclones were evaluated for Apaf-1 expression by immunoblot analyses (seebelow). The clones expressing high levels of the specific proteins were furthersubcloned by limiting dilution. Representative subclones of each of the HL-60transfectants were passaged twice per week and used for the studies. Datapresented are representative of those derived from at least two independentclonal transfectants of HL-60/Apaf-1 cells. Apaf-1-deficient MEFs (a gift fromDr. Tak W. Mak; The Amgen Institute, Toronto, Ontario, Canada) werecultured in DMEM (Life Technologies, Inc., Grand Island, NY) supplementedwith 10% fetal bovine serum (20). Apaf-11/2and Apaf2/2 MEFs weretransfected with 0.5–1.0mg of the appropriate plasmid DNA using the Lipo-fectAMINE PLUS reagent. Transfection efficiency was;40–60%, based oncotransfection of pCMV-GFP-1.

Western Analyses of Proteins.Western analyses of Apaf-1, Bcl-2, Bcl-xL,caspase-9, caspase-3, caspase-8, Bid, DFF, PARP, andb-actin were performedusing specific antisera or monoclonal antibodies according to previouslyreported protocols (25, 27, 28). Horizontal scanning densitometry was per-formed on Western blots by using acquisition into Adobe Photo Shop (Apple,Inc., Cupertino, CA) and analysis by the NIH Image Program (NIH, Bethesda,MD). The expression ofb-actin was used as a control.

Preparation of S-100 Fraction for the Analysis of Cytosolic Accumula-tion of Cyt c. Untreated and drug-treated cells were harvested by centrifuga-tion, and the cell homogenates were centrifuged at 100,0003 g for 30 min at4°C to obtain the S-100 fraction, as described previously. The supernatantswere collected, and the protein concentrations of S-100 were determined byusing the Bradford method (Bio-Rad, Hercules, CA). Samples were thenanalyzed for the release of cytc from the mitochondria into the cytosol byWestern blot, as described previously (29).

Apoptosis Assessment by Morphology and Annexin V Staining.Afterdrug treatment or the indicated transfections, 53 105 to 1 3 106 cells werewashed in PBS, and cytospin preparations of the cell suspension were fixedand stained with Wright stain. Morphological evaluation of apoptosis wasperformed as described previously (26). Cells were also resuspended in 100mlof staining solution (containing annexin V fluorescein and PI in a HEPESbuffer; annexin V-FLUOS staining kit; Boehringer Mannheim, Indianapolis,IN). After incubation at room temperature for 15 min, cells were analyzed byflow cytometry (25). Annexin V binds to those cells that express phosphoti-dylserine on the outer layer of the cell membrane, and PI stains the cellularDNA of those cells with a compromised cell membrane. This allows for thediscrimination of live cells (unstained with either fluorochrome) from apo-ptotic cells (stained only with annexin V) and necrotic cells (stained with bothannexin V and PI; Ref. 30).

Measurement of Mitochondrial Potential and ROS. To assess thechanges in mitochondrial potential and ROS, 13 10 6 cells were incubated for15 min at 37°C with 40 nM 3,39-dihexyloxacarbocyanine iodide (DiOC6; Ref.3) and 5mM dichlorodihydrofluorescein diacetate, respectively, and analyzedby fluorescence-activated cell sorting as described previously (31, 32).

RESULTS

Ectopic Overexpression of Apaf-1 Causes MitochondrialDCmand Cytosolic Accumulation of Cyt c. Stably transfected clones ofApaf-1-overexpressing (HL-60/Apaf-1) or control (HL-60/neo) cellswere isolated by limiting dilution (25) and examined for the process-ing of the caspases and death substrates as well as for the occurrenceof preapoptotic mitochondrial events (31, 32). Fig. 1A demonstratesdata from a representative clone of each cell type. As compared with

Fig. 1. Molecular events of apoptosis mediated by Apaf-1 in HL-60/Apaf-1versusHL-60/neo cells. Stably transfected Apaf-1-overexpressing cells and control (HL-60/neo)cells were harvested for Western analyses as well as flow cytometric analyses to deter-mine the percentage of cells with low MMP or increase in ROS (see “Materials andMethods”). A, inactive caspase-9 (p68) proform and its active cleaved fragment (Mr

;35,000), Zymogen procaspase-3 (p35) and its activated cleaved fragment (Mr ;17,000),PARP and itsMr 85,000 cleavage product, DFF45 and its intermediate cleaved product(Mr ;30,000), andb-actin, which was used as a control for equal loading.B, levels ofApaf-1, Bcl-2, andb-actin in HL-60/neoversusHL-60/Apaf-1 cells.C, cytosolic cytclevels in the S-100 fraction from HL-60/Apaf-1versusHL-60/neo cells. Increase in thepercentage of cells with reduced MMD (top panels) and increased production of ROS(bottom panels) in HL-60/Apaf-1versusHL-60/neo cells. Thearrow points to the positivecontrol after treatment with 10 mmol/liter H2O2.

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APAF-1-INDUCED ACTIVITIES AND CYTOSOLIC CYT c



HL-60/neo cells, HL-60/Apaf-1 cells showed 2.5-fold higher levels ofApaf-1 and demonstrated the processing and activation of pro-caspase-9 and procaspase-3 (Fig. 1,A and B). This was associatedwith the cleavage of PARP into itsMr 85,000 fragment and thecleavage of DFF45 (or ICAD) into itsMr 30,000 fragment. DFF45 andPARP are known substrates for caspase-3 (33, 34). As compared withHL-60/neo, Apaf-1 overexpression in HL-60/Apaf-1 cells was asso-ciated with a slightly increased expression of Bcl-2 (Fig. 1B) but notof Bcl-xL or Bax (data not shown). Whereas oligomerization ofApaf-1 is known to recruit, bind, and activate caspase-9 andcaspase-3, in HL-60/Apaf-1 cells, this was unexpectedly associatedwith a 5-fold increase in the cytosolic cytc (Fig. 1C), as well as anincrease in the percentage of cells with low MMP and increased ROS(Fig. 1D). These preapoptotic mitochondrial events and caspase acti-vations were also observed in HL-60 cells after transient transfectionand overexpression of Apaf-1 or the transfection of its longer, alter-nately spliced isoform (Apaf-1L), which possesses an additional WDrepeat (data not shown). Therefore, we used Apaf-1 transfectants inthe following experiments and refer to them henceforth as HL-60/Apaf-1 cells.

Ectopic Overexpression of Apaf-1 Induces Processing of Pro-caspase-8 and Bid and Sensitizes HL-60/Apaf-1 Cells to Etoposideand Paclitaxel. The activities of caspase-9 and caspase-3 can processand activate procaspase-8 and Bid; the latter, in turn, can cause themitochondrial release of cytc (4–6). Therefore we examined whetherApaf-1-mediated activities of the effector caspases would result in theprocessing of procaspase-8 and Bid, and whether this would sensitizeHL-60/Apaf-1 cells to etoposide- or Taxol-induced accumulation ofcyt c and apoptosis. As shown in Fig. 2,A andB, Apaf-1 overexpres-sion resulted in the processing of procaspase-8 and Bid, and, as notedpreviously, Bid processing caused cytosolic accumulation of cytc andapoptosis. Fig. 2Aalso shows that an exposure to Taxol (10 nM for24 h) or etoposide (1.0mM for 24 h) induced significantly moreapoptosis in HL-60/Apaf-1 cells than in HL-60/neo cells. Cotreatmentwith 50mM zVAD, which exerts more potent inhibitory effects againstthe activities of caspase-9 and caspase-3 than against the activities ofapical caspase-8 and caspase-10, also inhibited the processing ofcaspase-8 and Bid. Consequently, zVAD inhibited the cytosolic ac-cumulation of cytc and apoptosis induced by either Apaf-1 overex-pression or cotreatment with etoposide or Taxol (Fig. 2,A andB). Fig.

Fig. 2. zVAD blocks downstream events inducedby Apaf-1 overexpression in HL-60 cells. Stable HL-60/neo (control) or HL-60/Apaf-1 cells were treatedwith etoposide, Taxol, and/or zVAD for 24 h, and cellswere harvested to determine cell death (apoptosis andnecrosis) by annexin V/PI staining and protein levelsby Western analyses.A, the percentage of apoptotic(f)/necrotic (o) cells detected by annexin V/PI stain-ing. Error bars, SE for the mean percentage of apop-totic cells under the various treatment conditions.B,Western blot analyses of procaspase-8 (p55), Bid(p22), and its p15 cleaved product as well as thecytosolic levels of cytc. b-Actin was used as a controlfor equal protein loading.

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3 demonstrates that the mitochondrial release and cytosolic accumu-lation of cytc caused by Apaf-1 overexpression in HL-60/Apaf-1 cellsor caused by cotreatment with etoposide or Taxol in HL-60/neo cellsis also associated with reduced mitochondrial MMP (DCm) andincreased ROS. These mitochondrial perturbations in HL-60/Apaf-1cells, as in HL-60/neo cells, were also reversed by cotreatment withzVAD, indicating that these were secondary to the activities of theeffector caspases.

Transient Ectopic Expression of Apaf-1 Induces Apoptosis ofApaf-12/2 cells. We first compared the sensitivity of Apaf-12/2cells to a number of chemotherapeutic agents, including staurospo-rine, TNF-a, Fas ligation, and TRAIL. Fig. 4A demonstrates thatApaf-11/2 MEFs, which express Apaf-1 (Fig. 4B), were susceptibleto apoptosis induced by etoposide, doxorubicin, and staurosporine, butwere minimally susceptible to apoptosis induced by Taxol (1mM for24 h; Fig. 4A). Apoptosis of Apaf-11/2cells was also observed aftertreatment with TNF-a, Fas ligation, and TRAIL. In contrast, Apaf-12/2 MEFs lacking Apaf-1 (Fig. 4B) were resistant to etoposide,doxorubicin, and staurosporine-induced apoptosis (Fig. 4A). WhereasFas ligation and TNF-a were equally effective in inducing apoptosisin Apaf-11/2 and Apaf-12/2cells, TRAIL-induced apoptosis waspartially inhibited in Apaf-12/2cells (Fig. 4A). Etoposide-inducedprocessing of procaspase-9, procaspase-8, and Bid was comparedbetween Apaf-11/2and Apaf-12/2cells. Fig. 4Bdemonstrates that

etoposide-induced apoptosis of Apaf-11/2 cells is associated withthe processing of procaspase-9, procaspase-8, and Bid, whereas theseeffects of etoposide are absent in Apaf-12/2 cells. Transient trans-fection and overexpression of Apaf-1 cDNA increased the percentageof Apaf-11/2 MEFs demonstrating apoptosis. Apaf-1 transfectionalso sensitized Apaf-11/2cells to etoposide-induced apoptosis (Fig.5A). Transient transfection of Apaf-1 also induced apoptosis of Apaf-12/2 cells; Apaf-1 also sensitized these cells to etoposide-inducedapoptosis. Fig. 5Bshows that the transient transfection of Apaf-1 withor without etoposide resulted in the processing of procaspase-9, pro-caspase-8, and Bid.

Mitochondrial Perturbations Induced By Apaf-1 Overexpres-sion Are Caused Primarily by the Activities of Caspase-9 andCaspase-3 and not by Caspase-8 and Bid.We next determinedwhether the feedback effects on the mitochondria induced by Apaf-1overexpression were due primarily to the activities of caspase-9 andcaspase-3 or to the activities of processed caspase-8 and Bid. Tran-sient transfection and expression of DN caspase-9 (12), caspase-9b(18), or XIAP, which binds and inhibits caspase-9 and caspase-3 (35,36), markedly inhibited not only the processing of caspase-9 andcaspase-3 but also that of caspase-8 and Bid and also blocked mito-chondrial release of cytc and apoptosis (Fig. 6,A andB). In contrast,when procaspase-8 and Bid processing due to etoposide in HL-60/neocells or Apaf-1 overexpression in HL-60/Apaf-1 cells was directly

Fig. 3. zVAD reverses the decline in MMP (A) and increase in ROS (B) caused by the ectopic overexpression of Apaf-1 in HL-60/Apaf-1 cells (Apaf-1 ctr) or by treatment withetoposide or Taxol in HL-60/neo and HL-60/Apaf-1 cells. HL-60/neo or HL-60/Apaf-1 cells were treated with 10mM etoposide, 100 nM Taxol, and/or 50mM zVAD for 24 h. Flowcytometric analyses to determine the percentage of cells with either low MMP or increase in ROS were performed. InB, thedotted-line peak(indicated by anarrow) represents thepositive control after treatment with 10 mM H2O2. Thedashed-line peak(indicated by anarrowhead) represents the untreated HL-60/neo control or HL-60/Apaf-1 cells.

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inhibited by transient transfection of the cDNA of DN caspase-8 (37),there was minimal inhibition of the cytosolic accumulation of cytcand apoptosis (Fig. 7,A and B). In contrast, the processing of pro-caspase-8 (p55) and Bid was inhibited (Fig. 7B). Procaspase-3 proc-essing was not inhibited by DN caspase-8 in HL-60/neo or HL-60/Apaf-1 cells. Similarly, a transient transfection of the cDNA of CrmA(a cowpox virus encoded serpin-like protease inhibitor), which spe-cifically inhibits caspase-8 activity (5), also did not inhibit cytosolicaccumulation of cytc or apoptosis (data not shown). The dominantrole of the activities of caspase-9 and capase-3 over those of caspase-8followed by Bid in mediating the feedback effects on the mitochon-dria is further supported by the data presented in Fig. 8,A andB. Asshown, the effects of cotreatment with a relatively specific inhibitor ofthe activity of either caspase-9 (i.e., zLEHD-fmk) or caspase-8 (i.e.,

z-IETD-fmk) were compared (38). Again, inhibition of caspase-9 byz-LEHD-fmk inhibited the processing of procaspase-3, procaspase-8,and Bid and markedly inhibited the cytosolic accumulation of cytcand apoptosis induced by etoposide in HL-60/neo cells or by theectopic overexpression of Apaf-1 in HL-60/Apaf-1 cells. Inhibition ofcaspase-8 activity by 50mM z-IETD-fmk, in contrast, had a minimaleffect on the levels cytosolic cytc and apoptosis in both cell types.The difference in the pattern of the cleavage fragments of pro-caspase-3 in Fig. 8B versusFig. 7B may be due to the use oftetrapeptide inhibitors in the studies represented in Fig. 8B but not inthose represented in Fig. 7B. z-LEHD-fmk, but not z-IETD-fmk, alsoreversed the loss of mitochondrialDCm and the increase in ROSinduced by the ectopic overexpression of Apaf-1 in HL-60/neo cells(data not shown). The differential effect of treatment with z-LEHD

Fig. 4. Apaf-12/2MEFs are resistant to eto-poside-, Taxol-, doxorubicin-, staurosporine-, andTRAIL-induced apoptosis but not to TNF-a- andFas-induced apoptosis.A, Apaf-11/2 and Apaf-12/2 MEFs were treated with the indicated agentsfor 24 h, followed by staining with annexin V/PI todetermine apoptosis/necrosis.B, Western analysisof Apaf-1, procaspase-9, procaspase-8, Bid, andb-actin (or a loading control) in the Apaf-11/2(control) and Apaf-12/2MEFs treated with 100mM etoposide for 24 h.

Fig. 5. Apaf-1 induces apoptosis of the control Apaf-11/2cells and Apaf-12/2MEFs. Apaf-1 control cells and Apaf-12/2MEFs were transfected with the cDNA of Apaf-1for 24 h. Apaf-12/2cells transfected with vector (pcDNA) alone were also treated with 100mM etoposide for 24 h. After these transfections, cells were either (A) analyzed by annexinV/PI to measure apoptosis/necrosis or (B) harvested and analyzed for protein levels of Apaf-1, procaspase-9, and its cleaved fragments, Bid andb-actin, by Western analyses.

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versusz-IETD does not rule out the possibility that the observedmitochondrial events in HL-60/Apaf-1 cells, including the release ofcyt c, might also be due to the activation of other effector caspases, inaddition to caspase-3, by caspase-9.

DISCUSSION

In the present study, we demonstrate that the stable or transientoverexpression of Apaf-1 induces the processing of procaspase-9 and

procaspase-3 and causes the cleavage of the death substrates (PARPand DFF45) and apoptosis of the human acute myelogenous leukemiaHL-60 and murine Apaf-12/2cells. The cytosolic Apaf-1 binds andhydrolyzes ATP or dATP (11). Apaf-1 also binds to cytc (11, 12).The binding to cytc and the hydrolysis of dATP facilitate oligomer-ization of Apaf-1 (13–16). The multimeric Apaf-1-cytc complexbinds procaspase-9 with a stoichiometry of 1:1 (14). This results in theactivation and processing of caspase-9, which is then released fromthe Apaf-1-cyt c multimeric complex to process and activatecaspase-3 or other caspases including apical caspase-8 (15). There isalternative evidence that suggests that procaspase-9 can also recruitprocaspase-3 to the Apaf-1-procaspase-9 complex (16). Using a mu-tant procaspase-9 disabled for processing, it has also been demon-strated that caspase-9 can be activated without proteolytic processing(38). The spontaneously oligomerizing Apaf-530, which lacks theWD-40 domain, has been shownin vitro to bind and process pro-caspase-9 but lacks the ability to release the mature caspase-9 or torecruit procaspase-3 (15, 17). In contrast, in the data presented here,we demonstrate that the transient transfection of full-length Apaf-1 inHL-60 cells and in Apaf-12/2MEFs produces the processing andactivities of procaspase-9 and procaspase-3, resulting in apoptosis(25). However, these findings have to be regarded with the caveat thatthey were observed in an overexpression system, which may haveproduced an enforced oligomerization of Apaf-1, causing the proc-essing of caspase-9 and caspase-3 and apoptosis. As reported previ-ously, our data demonstrated that Apaf-12/2cells were resistant toapoptosis induced by chemotherapeutic agents, including etoposide,doxorubicin, and Taxol, but were not resistant to apoptosis induced byTNF-a or Fas ligation by an agonist antibody (20). In contrast,apoptosis induced by TRAIL was only partially inhibited in theApaf-12/2 cells. This may be because, as compared to Fas ligand orTNF-a, TRAIL-induced processing of procaspase-8 in Apaf-12/2MEFs may be modest and delayed due to the presence of DcR1 and/orDcR2 (39, 40), allowing Bid processing and the resultant mitochon-drial release of cytc. The presence of Bid in the cytosol clearly wouldnot facilitate caspase-9 and capase-3 activations in Apaf-12/2 cells(20, 21).

Our data show that Apaf-1 overexpression in HL-60 cells alsoresulted in the processing of procaspase-8 and Bid. This is most likelydue to the activity of caspase-9 and caspase-3, which can result in theprocessing of procaspase-8 (10, 22). Neither caspase-6 nor caspase-7has been shown to cleave Bid (41). In addition to the processing ofprocaspase-8 and Bid, ectopic overexpression of Apaf-1 was alsoassociated with the mitochondrialDCm and the release of cytc intothe cytosol. As shown in Fig. 9, these preapoptotic mitochondrialevents could be due directly to either the activities of caspase-9followed by caspase-3 or the intervening processing of procaspase-8and Bid. It should be noted that the inhibition of the activities ofcaspase-9 and caspase-3 by transient ectopic expression of caspase-9b, DN caspase-9, or XIAP (Fig. 6B) or by cotreatment with thecaspase-9-specific inhibitor zLEHD-fmk (Fig. 8B) markedly inhibitednot only the processing of procaspase-8 and Bid but also the preapo-ptotic mitochondrial release of cytc in HL-60/Apaf-1 cells. In con-trast, cotreatment with the caspase-8-specific inhibitor zIETD-fmk(Fig. 8B) or transient overexpression of DN caspase-8 (Fig. 7B) orCrmA (data not shown) did not inhibit the mitochondrial release of cytc. This indicates that the procaspase-8 and Bid processing mediated byectopic overexpression of Apaf-1 in HL-60 cells does not play adominant role in causing the feedback effects on the mitochondria.Therefore, the following question arises: how do caspase-9 and/orcaspase-3 (or another executioner caspase) activities triggered byectopic Apaf-1 mediate mitochondrialDCm and cytc release, if notthrough Bid activity? A recent report by Bossy-Wetzel and Green (41)

Fig. 6. DN caspase-9, caspase-9b, and XIAP inhibit Apaf-1- or etoposide-inducedcytosolic accumulation of cytc and apoptosis of HL-60 cells. Stably transfected HL-60/Apaf-1 and HL-60/neo (control) cells were transiently transfected with the cDNA of DNcaspase-9, caspase-9b, or XIAP for 24 h (see “Materials and Methods”). Cells weresubsequently harvested to determine (A) the percentage of apoptotic/necrotic cells byannexin-V/PI staining and (B) to perform Western analysis of procaspase-9, procaspase-8,procaspase-3, Bid, and its active cleaved product (Mr ;15,000) and cytosolic S-100fraction for cytc. b-Actin was used as a control for equal loading.

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suggested that the effector caspases such as caspase-3, caspase-6, andcaspase-7 that do not act directly on the mitochondria to release cytcmight cleave and activate another cytosolic substrate (other than Bid)that could then promote the mitochondrialDCm and the release of cytc. Our data also support the existence of such a putative cytosolic

MPTF. As shown in Fig. 9, MPTF may be the direct target forprocessing by caspase-9 and/or caspase-3 or caspase-7. Although Bidcleavage and activity triggered by death receptor signaling has clearlybeen shown to exert an amplifying role during apoptosis by causingmitochondrialDCm and cytc release, our findings suggest that this

Fig. 7. DN caspase-8 only partially inhibitsApaf-1- and etoposide-induced apoptosis and cytcrelease in HL-60 cells. Stable HL-60/neo and HL-60/Apaf-1 cells were transiently transfected with thecDNA of DN caspase-8 for 24 h. Subsequently, cellswere treated with etoposide, and the percentage ofapoptotic/necrotic cells was determined by annexinV/PI staining (A); cells were also harvested for West-ern analysis of procaspase-8, procaspase-3, Bid, andcytosolic cytc (B).

Fig. 8. As compared to the caspase-8 inhibitor z-IETD-fmk (IETD), the caspase-9-specific inhibitor, z-LEHD-fmk (LEHD), markedly inhibits the processing of procaspase-8,procaspase-3, and Bid and inhibits cytosolic accumulation of cytc and apoptosis induced by etoposide and the ectopic overexpression of Apaf-1. HL-60/neo and HL-60/Apaf-1 cellswere treated with 1.0mM etoposide and/or 50mM IETD or with 100mM LEHD for 24 h, and the cells were harvested for (A) annexin V staining to determine the percentage of apoptoticcells and (B) immunoblot analysis of procaspase-8 (p55), procaspase-3 (p35), Bid, and cytosolic accumulation of cytc. b-Actin levels were used as a control for protein loading.

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may not be the sole mechanism or may be part of the feedbackmechanism by which mitochondria are recruited for their role duringapoptosis.

Bcl-xL (or Bcl-2) has been shown to interact with Apaf-1 andinhibit Apaf-1-mediated activation of caspase-9 and caspase-3 andapoptosis (25, 42, 43). Boo, also identified as Diva, is a recentlydescribed, novel, antiapoptotic member of the Bcl-2 family. It formsa multimeric protein complex with Apaf-1 and caspase-9 (44, 45). Theexpression of Diva inhibited the binding of Bcl-xL to Apaf-1. Fur-thermore, Bak and Bik have been shown to disrupt the binding of Divato Apaf-1 (44). The inhibitor of apoptosis protein family of proteins,including XIAP, has also been shown to bind and inhibit caspase-9and caspase-3 (35, 36). Akt-1 kinase-mediated phosphorylation ofcaspase-9 appears to inhibit its activity and produce a similar effect(46). Taken together, these reports, along with the present data,suggest that a number of molecular determinants may regulate theApaf-1-mediated caspase activities, which could modulate the down-stream feedback activity of the MPTF on the mitochondria. Theimportance of the amplifying role of the putative MPTF for mito-chondrial input into apoptosis may vary according to the cellularcontext (47). Its importance is clearly suggested by the role thatApaf-1-mediated activation of caspase-9 and caspase-3 has been dem-onstrated to play during apoptosis and oncogenic transformation (24).Collectively, these aspects further underscore the greater complexityof the eukaryotic apoptosome as compared with theC. elegansmo-lecular machinery for apoptosis.

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2000;60:1645-1653. Cancer Res Charles L. Perkins, Guofu Fang, Caryn Nae Kim, et al. or Paclitaxel-induced Mitochondrial Events during ApoptosisThe Role of Apaf-1, Caspase-9, and Bid Proteins in Etoposide-

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