Article |
Address correspondence to David Cheresh, The Scripps Research Institute, Department of Immunology, IMM24 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: (858) 784-8281. Fax: (858) 784-8926. E-mail: cheresh{at}scripps.edu
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Abstract |
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Key Words: cell adhesion; apoptosis; caspase; integrin; ligands
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Introduction |
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Unlike cytokine receptors, however, integrins function selectively as "solid state" receptors (Ingber, 1992). Whereas a substrate-immobilized ligand can serve as an integrin agonist, the same ligand in solution functions as an integrin antagonist. This characteristic of the integrin may relate to an altered capacity of the integrins to cluster (Broday, 2000), recruit kinases such as focal adhesion kinase (Hildebrand et al., 1993), tether adaptor proteins and cytoskeletal elements (Wary et al., 1996), and/or provide mechanical tension (Hocking et al., 2000). Irrespective of the mechanism, it is clear that both of these distinct integrin states provide information to the cell about the nature of its environment.
For example, de novo expression of integrin vß3 occurs on endothelial cells in response to angiogenic growth factors (Brooks et al., 1994a), allowing increased endothelial cell interaction with the deposited provisional ECM. Soluble antagonists of this receptor initiate endothelial cell apoptosis, suppressing angiogenesis in various animal models (Brooks et al., 1994b; Storgard et al., 1999). Notably, cell death does not result from the loss of adhesion, per se, as observed during anoikis (Frisch and Francis, 1994). Instead, apoptosis results from the selective blockade of a single integrin, in this case
vß3, on adherent, tissue-associated cells. Similar observations have been made involving other integrins. Notably, the overexpression of unligated integrin
5ß1 has been associated with apoptosis and reduced tumor cell growth in vitro and in vivo (Giancotti and Ruoslahti, 1990; Varner et al., 1995; Kim et al., 2000; Plath et al., 2000). These observations suggest that the expression of specific integrin complexes, in the absence of a suitable ligand, may promote death. Physiologically, this would provide a mechanism whereby cells that find themselves within an inappropriate ECM would be actively cued to undergo apoptosis.
To understand how integrins, in the ligated or unligated state, influence cell survival in a physiological environment, cells with a defined integrin profile were evaluated in the context of a homogenous three-dimensional ECM. The evidence provided demonstrates that simple expression of unligated integrin is sufficient to initiate cell death among adherent cells. This "integrin-mediated death" (IMD) was similarly induced by the cytoplasmic domain of ß1 or ß3 integrins, resulting in the recruitment of caspase-8 to the cell membrane and its subsequent activation. These results reveal an unexpected role for integrins as proactive mediators of cell death, and document a novel mechanism for the induction of apoptosis during tissue remodeling and homeostasis.
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Results |
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Endothelial cells survive when cultured in fibrin, an ECM that ligates integrin vß3 and at least two ß1 integrins, yet undergo apoptosis when cultured in collagen (Fig. 2 A) (Filardo et al., 1995). Similarly, endothelial cells undergo apoptosis when cultured on the surface of collagen gels, but not on fibrin gels (Fig. 2 A, open bars). The induction of death was not simply due to an inability to attach and spread on collagen, since endothelial spreading on collagen and fibrin occurred to the same degree and with similar kinetics (Fig. 2 B). However, endothelial cells cultured on collagen were prone to apoptosis, leading to laemelopod retraction, blebbing, nuclear condensation, and eventual detachment.
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The cytoplasmic domain of the integrin ß subunit is proapoptotic
Integrin-mediated signaling depends largely on the cytoplasmic domains of the and ß subunits. To determine whether the integrin cytoplasmic domain was sufficient to induce death, chimeric proteins composed of the extracellular domain of CD25 (IL2Ra, Tac) and the cytoplasmic domains of either integrin
5, ß1, or ß3 (LaFlamme et al., 1992) were expressed in COS7 cells. The expression of either Tac-ß1 or Tac-ß3 constructs resulted in increased death, while expression of the
5 chimera did not (Fig. 3 A), despite similar expression levels (Fig. 3 B). Tac-ß3 expression produced a dose-dependent death among attached cells (Fig. 3 B). Importantly, the expression of these integrins was similar to, or less than, that of native integrins. Death occurred via apoptosis, as indicated by annexin-V reactivity (Fig. 3 C, top) and by processing of the caspase substrate, poly (ADP-ribose) polymerase (PARP), to the characteristic 85-kD apoptotic fragment (Fig. 3 C, bottom). IMD was induced efficiently by Tac-ß3 or Tac-ß1, yet only weakly by Tac-ß5 (Fig. 3 D).
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The expression of GFP-ß3 also allowed integrin-transfected cells to be monitored to assess whether cell spreading was compromised. In agreement with the previous endothelial cell spreading studies (Fig. 2 B), no difference in cell spreading was observed between GFP-ß3 and control GFP-expressing cells until after apoptosis was evident (Fig. 4 F). Thus, the onset of apoptosis can initiate cell retraction and eventual detachment (Harrington et al., 2001).
Integrin-mediated substrate attachment, accomplished by adhesion to ECM proteins (Ilic et al., 1998; Scatena et al., 1998) or by integrin binding to substrate-immobilized antibodies (Stromblad et al., 1996), suppresses apoptosis. In contrast, antagonized (Brassard et al., 1999) and/or nonligated integrins (Stromblad et al., 1996; and this manuscript) promote apoptosis among otherwise adherent cells. To evaluate whether the integrin chimeras would promote death when substrate-immobilized, Tac-ß3expressing COS7 cells were replated on surfaces coated with an anti-Tac monoclonal antibody. This attachment and cell spreading enriched the number of integrin chimeraexpressing cells (Fig. 4 G, top), yet these cells demonstrated a dramatic reduction in apoptosis (60% decrease in PARP cleavage) relative to the adherent control population (Fig. 4 G, bottom). In other experiments, we also tested whether cells treated with soluble anti-Tac or anti-Tac on microbeads could prevent IMD, but neither impacted this event (unpublished data). Our result suggests that only substrate-localized integrin "clustering" was capable of suppressing the death-promoting activity of the ß3 tail.
IMD requires initiator caspases
To begin to address the molecular mechanism responsible for IMD, checkpoint-specific inhibitors of caspase activation were expressed in COS7 cells undergoing IMD. Expression of dominant negative caspase-9 or bclxl, which inhibit the stress-mediated caspase cascade (Finucane et al., 1999; Soengas et al., 1999; Wolf and Green, 1999), did not prevent IMD, but did block stress-induced apoptosis of these cells (Fig. 5 A). In contrast, crmA, a serpin that inhibits activation by death receptoractivated caspases (Zhou et al., 1997), protected cells from IMD (Fig. 5, A and B). Surprisingly, a negative-acting variant of the death receptor adaptor, FADD (Fas-associating protein with death domain), which inhibits Fas-initiated apoptosis mediated by death receptors (Chinnaiyan et al., 1996), was not protective (Fig. 5, A and B), despite the fact that it effectively blocked the Fas-mediated death of these cells (Fig. 5 A). Apoptosis could also be prevented by treatment of integrin tailexpressing cells with peptide inhibitors of initiator and executioner caspases (Fig. 5 C) (Thornberry et al., 1997), but was unaffected by a stress caspaseselective inhibitor (zLEHDfmk).
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Unligated integrins activate caspase-8like activity
To explore the mechanism(s) by which unligated integrins might activate caspase and induce IMD, we next examined the subcellular localization of activated caspases during IMD. Integrin-initiated caspase activity was visualized by labeling with FAM-VADfmk, which covalently and fluorescently labels activated caspases. Visual assessment of cells undergoing IMD revealed a punctate distribution of caspase-associated fluorescence that colocalized with unligated ß3 integrin tail on the cell surface (Fig. 6 A). In contrast, Tac-5, which does not induce IMD, appeared diffusely on the cell surface and did not colocalize with caspase activity (Fig. 6 A). Notably, caspase activity was also found to be reduced in or absent from Tac-ß3expressing cells that were attached to substrate-immobilized Tac antibody (Fig. 6 A, left). In these cells, Tac-ß3 expression was found to be redistributed and predominantly at the cellsubstrate interface, as determined by confocal z-section analysis. The series of collected, integrin-containing z-sections were subjected to digital image analysis by blinded observers to assess possible colocalization between the integrin and active caspase (Fig. 6 B). Only under conditions that induced IMD (unligated Tac-ß3), was significant colocalization observed between caspase and integrin (coefficient of colocalization = 0.49 ± 0.07; P < 0.01).
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Recruitment of caspase-8 by integrin ß subunit tails
The external clustering of death receptors, such as Fas, leads to formation of a death-inducing signaling complex (DISC), which recruits and activates caspase-8 (Scaffidi et al., 1999). To determine if integrins induced apoptosis in a manner analogous to death receptors, COS7 cells were held in suspension and treated with anti-vß3 integrinantagonist antibodies. The cells were lysed and integrin complexes were then immunoprecipitated from the resulting lysates and subjected to analysis for DISC components. Although both the zymogen and active forms of caspase-8 were observed in integrin immunoprecipitates (Fig. 7 A), FADD was not detected (unpublished data). Clustering of a control transmembrane protein, LDL-like receptor protein (LRP), which is expressed at levels similar to integrin
vß3 on COS7 cells, did not recruit either form of caspase-8 (Fig. 7 A).
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To determine whether endogenous integrins induced IMD by recruitment of caspase-8, native vß3 complexes were isolated from endothelial cells undergoing apoptosis in collagen gels and assessed for the presence of caspase-8. Endothelial cells cultured in collagen or in suspension (positive apoptosis control) were enriched for the presence of caspase-8 (Fig. 7 D), whereas complexes isolated from endothelial cells cultured in a fibrin gel displayed a dramatic decrease in both caspase-8 (Fig. 7 D) and apoptosis (Fig. 2 A). Thus, cells expressing high levels of endogenous
vß3, in an inappropriate ECM, can be induced to die by the death receptorindependent, and presumably, FADD-independent, recruitment and activation of caspase-8. Conversely, this process is prevented in the presence of an integrin-appropriate ECM.
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Discussion |
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IMD
IMD depends upon the ability of the integrin ß subunit to recruit caspase-8 to the cell surface in a FADD-independent manner. These findings may explain our previous observations in vivo, where angiogenesis was suppressed in tissues treated with vß3 integrin antagonists due to an induction of apoptosis of angiogenic endothelial cells (Brooks et al., 1994b; Stromblad et al., 1996; Storgard et al., 1999). In recent studies, we observed that inhibitors of caspase-8, but not caspase-9, restored neovascularization in
vß3 antagonisttreated angiogenic tissues (unpublished data). Thus, cells that interact with an inappropriate ECM, or those exposed to integrin antagonists may succumb to IMD. Interestingly, while ß1 and ß3 integrins can promote this process, ß5 apparently does not (Fig. 3 D). Therefore, distinct integrins may have different capacities to induce apoptosis due to regulatory regions present elsewhere in the integrin heterodimer.
IMD was observed in several cell lines, including COS7, CS1, HEK, HTB, and HUVEC. However, it is noteworthy that some cells examined, including CHO-K1 and HeLa, were resistant to IMD (unpublished observations). Resistance to IMD may occur, in vivo or in vitro, for several reasons. Clearly, cell typespecific or clonal variations result in cultured cells having varied capacities to secrete or otherwise remodel their immediate ECM. Furthermore, not all cells express appropriate caspases, such as caspase-8, or may express inhibitors such as c-flip (Aoudjit and Vuori, 2001). In these cases, integrin antagonism may induce apoptosis by other means. For example, integrin antagonism also leads to the activation of the tumor suppressor p53 (Stromblad et al., 1996). Since p53 complexes with caspase-8 (Ding et al., 2000), it may have a direct role in IMD. However, transcriptional activation of p53 also elicits stress-mediated death via activation of caspase-9 (Soengas et al., 1999). Accordingly, neurons that express little or no caspase-8 eventually undergo apoptosis in response to integrin antagonism via the stresscaspase-9 pathway (Bonfoco et al., 2000).
In addition, a variety of other proteins that can play key regulatory roles in the induction of, or resistance to, apoptosis have been identified. For example, tumor cells that are anchorage independent may be resistant to IMD. However, the expression of integrin vß3 on CS1ß3 cells was able to induce IMD in an otherwise anchorage-independent cell (Fig. 1 D). These findings suggest that specific integrin antagonists may be useful as antitumor agents, as suggested by others (Ruoslahti, 1997; Curley et al., 1999).
Physiological roles for IMD
IMD would be predicted to occur during tissue remodeling associated with development, wound repair, or inflammation to ensure that only appropriate cells remain viable within a given tissue microenvironment. For example, the ECM is dramatically remodeled during the involution/epithelial cell regression phase following weaning (Sympson et al., 1994). During this apoptotic process, epithelial cells undergo caspase activation in a ß1 integrindependent manner (Boudreau et al., 1996). Antagonism of ß1 also results in chondrocyte apoptosis during ECM remodeling associated with differentiation (Hirsch et al., 1997). In agreement with these studies, we show that the cytoplasmic domain is sufficient to induce IMD. However, it is not clear why apoptosis is more often associated with specific ß1 integrins. In particular, the overexpression of integrin 5ß1 has been documented to suppress tumorigenicity (Giancotti and Ruoslahti, 1990; Varner et al., 1995) and/or angiogenesis (Kim et al., 2000), possibly through the induction of apoptosis (Plath et al., 2000). The effect is reversed by fibronectin, a ligand for
5ß1 (Varner et al., 1995). In contrast, proliferative tumors with an elevated expression of
5ß1 in vivo, which would be IMD resistant, might therefore be associated with a poorer prognostic outcome (Adachi et al., 2000). Thus, IMD may be an important regulatory event under many physiological circumstances in vivo.
Role of caspase-8 in IMD
Our findings indicate that caspases function downstream of unligated integrins in the induction of IMD. Thus, inhibition of caspase-8 via peptide or protein (crmA) antagonists results in reduced IMD, whereas increased expression of unligated integrins increases caspase activation and apoptosis. Evidence is provided that caspase-8 can directly or indirectly interact with integrin ß subunit cytoplasmic domain, and this interaction contributes to the activation of the caspase. Unligated integrins tend to cluster on the cell surface in a manner dependent upon integrin expression level, however, these integrins are not associated with focal adhesion kinase or tethered by the cytoskeleton (unpublished data). These untethered clusters of integrin may serve to recruit sufficient numbers of caspase-8 zymogens to facilitate proximity-induced activation of the initiator caspase cascade (induced proximity model; Salvesen and Dixit, 1999) in a death receptorindependent manner. Although integrins may be less efficient than death receptors in this respect, it is noteworthy that other methods of "clustering" caspase-8 serve to activate it, including mutation, to add multimerization domains (MacCorkle et al., 1998; Memon et al., 1998; Muzio et al., 1998; Fan et al., 1999), and overexpression strategies (Stennicke and Salvesen, 1999). Supporting this notion, integrin cytoplasmic domain constructs that provide no localization signal, e.g., His-ß3, also fail to induce IMD, whereas, increasing the number of unligated integrins (Figs. 1 B and 3 B) or blocking multiple integrins (vß3 and ß1 integrins; Fig. 2 F), augmented the incidence of IMD. Thus, the increased apoptotic rate seen with combined anti-
vß3/anti-ß1 treatment in collagen gels (Fig. 2 F) may be due to the increased sum quantity of unligated integrins (in this case, both ß3 and ß1).
Integrin- or caspase-deficient mice frequently show developmental defects and/or lethality (Fassler et al., 1996; Zheng and Flavell, 2000). Although vß3 is proapoptotic when unligated (this work), or antagonized (Brooks et al., 1994b; Brassard et al., 1999; Storgard et al., 1999), it is, nonetheless, nonessential for the viability of humans (Coller et al., 1991) or mice (Hynes and Hodivala-Dilke, 1999). Why then, should
vß3 continue to be expressed on invasive tissues in the broad range of species observed? Integrin
vß3 may function during invasive processes as a biosensor, providing positive feedback to the cell during "productive" interactions, i.e., in a permissive ECM (Ilic et al., 1998), while inducing death by triggering caspase recruitment/activation in those cells that enter an inappropriate microenvironment. Disrupting expression of integrin
vß3 would thus only remove one of several possible triggers for apoptosis. This capacity to selectively suppress or promote apoptosis distinguishes integrins from the death receptor and stress apoptotic pathways, and suggests that integrins may be grouped within a third emerging category of apoptotic triggers, the dependence receptors (Bredesen et al., 1998).
It is still not known whether accessory molecules may be involved in the regulation of this interaction. However, not all cells were subject to IMD, and not all integrins are proapototic. Thus, it is likely that extended investigations will reveal both cell and integrin-specific factors that govern the induction of this cascade. Nevertheless, this novel apoptotic trigger, which is regulated by integrin repertoire and local ECM composition, provides a new means to account for cell death and survival associated with tissue remodeling and homeostasis.
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Materials and methods |
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Constructs and gene delivery
Deficient adenoviral antisense constructs containing a CMV-driven ORF complementary to the first 508 bp of ß3 or nonsense (empty read through) constructs were generated by recombination of pAdCI/pJM17 plasmids in U293 (E1 complementary) cells. HUVECs were infected at a multiplicity of infection of 100 pfu/cell, yielding 100% infection as assessed by infection with adenoviruses encoding an Ad-ß gal reporter gene. The pCI-NeoTac-ß3, pCI-NeoTac-ß5, and pCI-NeoTac-ß1 constructs were created by ligation of integrin cytoplasmic domains to the extracellular and transmembrane domains of Tac/CD25, as originally reported (LaFlamme et al., 1992). The truncated chimeras were created by introduction of inframe stop codons in the integrin coding sequence, as described (Filardo et al., 1995). The His-ß3 construct was constructed by in frame expression of the ß3 cytoplasmic domain after the His tag in pcDNA3.1/V5/His A vector (Invitrogen). The GFP-ß3 chimeras were constructed essentially as described for ß1 (Smilenov et al., 1999) but included the signal sequence and transmembrane/cytoplasmic domains of integrin ß3, rather than ß1, expressed in the pEGFP-C3 vector (CLONTECH Laboratories, Inc.). The bclxl, bad, and crmA constructs in pCMV5 were the gifts of S. Huang (The Scripps Research Institute, La Jolla, CA), independent DN-FADD constructs that lack the DED domains were obtained from H. Duan (University of Michigan, Ann Arbor, MI) and E. Li (The Scripps Research Institute, La Jolla, CA). DNcaspase-9 and Fas were provided by G. Salvesen (The Burnham Institute, La Jolla, CA). For the induction of death by chimeras, subconfluent (
30% confluent) cell cultures were transfected via lipofectamine or lipofectamine plus, as per the manufacturer's instructions (Invitrogen), and harvested at the time points described. For PARP cleavage studies, cells were lysed on the plate in complete RIPA. For PI studies, cells were briefly trypsinized and collected. Cells were labeled with Alexa-488conjugated anti-CD25 (7G7B6), and PI was added to assess loss of viability. To avoid misinterpretation of DNA toxicity in coexpression experiments, only 2 µg of pCI-NeoTac-ß3 was cotransfected with 4 µg of the specific apoptotic inhibitor, as described in the cotransfection/rescue studies. Expression of these constructs was confirmed by FACS® and/or immunoblotting, as described in the legends.
Assessment of apoptotic indicators
For FACS® analysis of apoptosis, only cells remaining adherent on tissue culture plastic after washing with PBS were trypsinized briefly, quenched in complete media, and again washed in PBS, before annexin-V staining and assessment on a FACScan® flow cytometer (Becton Dickinson). Tac-expressing cells (Alexa 594, FL3) were identified as apoptotic by annexin staining (FITC, FL1). For PARP cleavage studies, cells were lysed in RIPA (Filardo et al., 1995), cell debris was pelleted at 16000 g, and lysates were analyzed by immunoblotting (2 µg/ml PARP; catalog number 66391A; PharMingen), 5 µg/ml Tac mAb 7G7B6 (American Type Culture Collection), rabbit antisera to GFP (Santa Cruz Biotechnology, Inc.) via species-specific secondary HRP-conjugated antiserum (Jackson ImmunoResearch Laboratories) and Lumigen PS-3 improved chemiluminescent substrate (Lumigen). The 115- and 85-kD caspase-processed forms were quantitated (IP Lab Spectrum Software) within each experiment. Data was expressed as a ratio of p85/p115 for the lane (construct expressing and nonexpressing cells, inclusive). Caspase activity isolated from total cell populations was also assessed using LEHD-pNA (unpublished data) and IETD-pNA colorimetric assays according to the manufacturer's protocol (Chemicon).
Cell spreading
Digital images of cells were captured serially during spreading using a Princeton Instruments Micromax CCD camera (Roper Scientific, Inc.). Cell spreading was quantified by segmentation of GFP or GFP-ß3expressing cells (COS7) or Cell Tracker-Greenlabeled cells (HUVEC) with automated area calculations using IP Lab Spectrum Software (Scanalytics). The average cell spreading was determined from the cell area on 10 random fields for each time point.
Adhesion to antibody-coated substrate
Anti-Tac (5 µg/ml in PBS, pH 8.1) was allowed to coat nontissue culture dishes or coverslips overnight at 4°C. Surfaces were washed twice with PBS before cell adhesion. COS7 cells expressing Tac 12 h after transfection with 40 ng Tac-ß3 pCI-Neo/cm2 were trypsinized, quenched, and allowed to seed the anti-Taccoated plates in serum-free media. After 1 h at 37°C, unattached cells were removed by washing with serum-free media, and complete media was added. Cells that were lysed and analyzed for integrin expression and PARP cleavage (as described) were compared 24 h later to control populations still adherent on their original 100-mm dish.
Membrane isolation and immunoprecipitations
Cellular fractionation was performed as previously described (Chen et al., 2000). Pelleted membranes were resolubilized in Laemmli buffer for gel analysis, or in 1x protease inhibitor cocktail (1% Triton X-100, 25 mM Hepes, pH 7.4; Boehringer) for immunoprecipitation with anti-GFP agarose (Santa Cruz Biotechnology Inc.). Alternately, integrincaspase complexes were immunoprecipitated by adding precipitating antisera (polyclonal anti-ß3, polyclonal anti-GFP, or polyclonal anti-LRP) to form DISC-like structures, followed by lysis on ice in 1% triton, as described (Scaffidi et al., 1999). Caspase immunoblotting was performed with anticaspase-8 polyclonal (Chemicon; StressGen Biotechnologies) or monoclonal (Calbiochem; mAb3) antibodies. These antibodies recognize both the zymogen and active forms of the caspase. The potential presence of other caspases was excluded by immunoblotting for the presence of caspases-1, -3, -6, -7, -9, and -10 using specific rabbit polyclonal antisera (Chemicon; StressGen Biotechnologies) that recognize both zymogen and active forms of the caspase. Even though caspases-3, -8, and -9 were the predominant caspases in COS7 cells, all but caspase-1 and -10 were detected.
Caspase labeling and inhibition with peptides
The methyl esterderived peptide inhibitors zIETDfmk, zDEVDfmk, and zLEHDfmk (Calbiochem) or zVADfmk (Bachem) were reconstituted in DMSO (50 mM) and diluted as appropriate. Apoptosis was assessed in subconfluent 33-mm dishes of COS7 cells expressing submaximal quantities of death-inducing constructs (12 µg of Tac-ß3/pCI-NEO) using 4080 µM peptides (or DMSO diluent) by visual scoring (not shown) and via the cleavage of the executioner caspase substrate PARP. Fluorescent FAM-VAD (InterGen) labeling of caspase in Tac-expressing cells was performed according to the manufacturer's specifications, followed by fixation (PBS/1% paraformaldehyde) and blocking (PBS/3%BSA) for 1 h at 37°C, and detection of Tac with Alexa-594conjugated (Molecular Probes) mAb 7G7B6 (0.5 µg/ml) for 1 h. Cells were washed three times with PBS and examined with a Bio-Rad Laboratories 1024 confocal microscope. Biotinylation of active caspases with biotin-VADfmk was performed by first removing serum-containing media, washing the cells once, and incubating at a final concentration of 10 µM biotin-VAD in DME for the final hour. Biotinylated complexes were isolated with avidin-Sepharose, identified by probing with antibiotin horseradish peroxidaseconjugated monoclonal antibody (Sigma-Aldrich), and developed via CDP-star reagent (Applied Biosystems). Membranes were subsequently reprobed with caspase-specific antisera (-3, -6, -7, -8, -9) as described.
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Footnotes |
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Acknowledgments |
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This work was supported by grants CA45726, CA50286, and CA78045 (D.A. Cheresh) and AR02089 (C.M. Storgard) from the National Institutes of Health. X.S. Puente is a recipient of a Human Frontier Science Program fellowship. This is Scripps Research Institute manuscript number 12974-IMM.
Submitted: 14 June 2001
Revised: 18 September 2001
Accepted: 19 September 2001
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