Apoptosis-associated caspase activation assays

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Methods 44 (2008) 262–272 www.elsevier.com/locate/ymeth

Apoptosis-associated caspase activation assays

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Scott H. Kaufmann a,b,*, Sun-Hee Lee b, X. Wei Meng a,b, David A. Loegering a, Timothy J. Kottke a, Alexander J. Henzing c, Sandrine Ruchaud c, Kumiko Samejima c, William C. Earnshaw c b

a Division of Oncology Research, Mayo Clinic College of Medicine, 200 First Street, S.W., Rochester, MN 55905, USA Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, MN 55905, USA c Institute of Cell & Molecular Biology, University of Edinburgh, Edinburgh, EH9 3JR Scotland, UK

Accepted 10 November 2007

Abstract Caspases are aspartate-directed cysteine proteases that cleave a diverse group of intracellular substrates to contribute to various manifestations of apoptosis. These proteases are synthesized as inactive precursors and are activated as a consequence of signaling induced by a wide range of physiological and pathological stimuli. Caspase activation can be detected by measurement of catalytic activity, immunoblotting for cleavage of their substrates, immunolabeling using conformation-sensitive antibodies or affinity labeling followed by flow cytometry or ligand blotting. Here we describe methods for each of these assays, identify recent improvements in these assays and outline the strengths and limitations of each approach.  2007 Elsevier Inc. All rights reserved. Keywords: Caspases; Apoptosis; Affinity labeling; Enzymatic activity; Fluorogenic substrate; Flow cytometry; Immunoblotting; Immunofluorescence

1. Introduction Caspases are a family of intracellular cysteine proteases. Caspase 1, the founding member of the family, was originally identified based on its ability to cleave pro-interleukin-1b to the mature cytokine. Subsequent studies identified caspases 2–14 (numbered in order of cloning) and demonstrated roles for caspases 2, 3, 6, 7, 8, 9, and 10 during apoptosis [1]. Biochemical and crystallographic analyses have demonstrated that members of the caspase family share several features, including a classical histidine/cysteine catalytic dyad and a strong preference for

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Supported in part by a Grant from the NIH (R01 CA69008). S.-H.L. is a recipient of a studentship from the Mayo Foundation. W.C.E. is a Principal Fellow of the Wellcome Trust. * Corresponding author. Address: Division of Oncology Research, Mayo Clinic College of Medicine, 200 First Street, S.W., Rochester, MN 55905, USA. Fax: +1 507 284 3906. E-mail address: [email protected] (S.H. Kaufmann). 1046-2023/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2007.11.005

cleavage at the C-terminal side of aspartate [1,2] or, in some cases, glutamate [3,4] residues. Each caspase gene encodes a precursor that contains a prodomain, a large subunit and a small subunit. Based on their functions, apoptotic caspases are now classified as initiator caspases, which transduce various signals into proteolytic activity, and effector caspases, which cleave most of the more than 400 substrates that are degraded in cells undergoing apoptosis [1,5,6]. These two classes of caspases differ in not only their functions, but also their quaternary structure, requirements for activation, and abundance. In their zymogen forms, initiator caspases are present as monomers in the cytoplasm [7]. There are two classical pathways for activating these proteases. In the extrinsic pathway, ligation causes cell surface receptors such as CD95/Fas to bind and oligomerize the cytoplasmic adaptor molecule FADD [8]. The subsequent binding of the prodomains of procaspases 8 and/or 10 to FADD leads to presumed oligomerization of these procaspases [9],

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causing a conformational change that results in acquisition of enzymatic activity [7,10]. In the intrinsic pathway, release of cytochrome c from mitochondria results in dATP- or ATP-dependent oligomerization of the cytoplasmic scaffolding molecule Apaf-1 (apoptotic protease activating factor-1), which in turn binds and presumably oligomerizes the procaspase 9 zymogen [9,11], leading to a conformational change at its active site that results in acquisition of enzymatic activity [12]. Procaspases 8, 9 and 10 in turn appear to participate in activation of downstream effector caspases, which exist as inactive zymogen dimers within the cytoplasm [7]. Effector caspases are activated by proteolytic cleavages at specific aspartate residues [1,13]. These cleavages separate the large and small subunits from each other [13], while simultaneously causing a conformational change that displaces a peptide loop that occludes the active site of the inactive effector caspase zymogens [14]. One current model suggests that caspases 8, 9, and 10 are able to proteolytically activate procaspases 3 and 7, which are in turn responsible for proteolytically activating procaspase 6 [1]. Alternative activation schemes, however, have also been proposed. For example, caspase 1 reportedly activates caspase 6 to initiate apoptosis in neurons [15]. Other caspases appear to participate in apoptosis following specific stimuli. Procaspase 2 has recently been reported to be the initiator caspase when cells respond to heat shock [16]. Once activated, caspase 2 cleaves and activates the proapoptotic Bcl-2 member Bid, thereby triggering the intrinsic pathway, with release of cytochrome c, activation of caspase 9, and subsequent activation of effector caspases [17]. At present, the biochemical basis for the heat shock-induced activation of the caspase 2 zymogen remains to be more fully defined. Because various initiator caspases are activated by different signals, identifying the caspases that are activated and their order of activation after treatment of cells with a particular stimulus can provide insight into the lethal signaling that is induced by that stimulus. Toward this end, the caspase activation process can be followed using assays for cleavage of suitable fluorogenic or chromogenic substrates, immunoblotting with monospecific antibodies, immunochemistry with conformation-sensitive antibodies, or affinity labeling with reactive substrate analogs. In the sections that follow, we outline these various approaches, describe recent improvements in these methods, and review the strengths as well as potential limitations of some of the assays. 2. Caspase activity assays 2.1. Overview Conceptually, the most straightforward method for determining whether caspases have been activated in apoptotic cells is to assay for their ability to cleave model substrates. This approach became feasible after the

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identification of sequences that are cleaved in various caspase substrates during apoptosis [reviewed in 1] and the development of synthetic substrates containing the appropriate peptides coupled to fluorogenic or chromogenic leaving groups [13,18]. Because caspases are intracellular proteases, lysates or cell fractions must first be prepared, then assayed for protein content, and finally examined for ability to cleave fluorogenic or chromogenic substrates. Ideally, the product produced should be compared to a standard curve so that activity can be reported in a reproducible fashion. 2.2. Chemicals and reagents The peptide-coupled aminofluorocoumarins N-acetylaspartyl-glutamylvalinylaspartyl-7-amino-4-trifluoromethylcoumarin (DEVD-AFC) and N-acetylvalinylglutamylisoleucylaspartyl-AFC (VEID-AFC) were from Biomol (Plymouth Meeting, PA). An Apo-ONE homogenous caspase-3/7 kit, which is based on the liberation of free rhodamine 110 from peptide-bound substrate, was obtained from Promega (Madison, WI). Unconjugated AFC, etoposide and cytarabine were from Sigma (St. Louis, MO). Unconjugated rhodamine 110 and 17-AAG are available from Invitrogen Molecular Probes (Eugene, OR) and Calbiochem (San Diego, CA), respectively. All other chemicals were reagent grade. For the examples provided below, parental and caspase 6 / DT40 chicken lymphoma cells [19] as well as ML-1 human leukemia cells [20] were cultured and treated with apoptotic stimuli as previously described. 2.3. Preparation of subcellular fractions Because peptide-based caspase substrates are poorly cell permeable, cells must typically be lysed before the assay is performed. In the example shown in Fig. 1A, 108 cells were sedimented at 200g. All further steps were performed at 4 C. Cells were washed twice in calcium- and magnesium-free Dulbecco’s phosphate-buffered saline (PBS), then resuspended at a concentration of 1–3 · 108 cells/ml in buffer A, which consists of 25 mM HEPES (pH 7.5 at 4 C), 5 mM MgCl2, and 1 mM EGTA supplemented immediately before use with 1 mM a-phenylmethylsulfonyl fluoride (PMSF), 10 lg/ml pepstatin A, and 10 lg/ml leupeptin. Following a 20-min incubation, cells were subjected to 20–50 strokes in a tight-fitting Dounce homogenizer. After disruption of the plasma membrane was confirmed by phase contrast microscopy, samples were sedimented at 800g for 10 min. The postnuclear supernatant was then mixed with 1/100th volume of 0.5 M EDTA and sedimented at 280,000gmax for 60 min in a Beckman TL-100 ultracentrifuge. Aliquots of the supernatant from this spin (cytosol) were used for protein determination [21] or supplemented with dithiothreitol to a final concentration of 1 mM and frozen at 80 C for up to 3 months until activity was determined. In prior studies, caspase

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Fig. 1. Measurement of caspase activity using a fluorogenic substrates. (A) After parental DT40 chicken lymphoma cells and a caspase-6 / clone [19] were treated with etoposide for 5 h, cytosol was prepared [23]. Aliquots containing 50 lg of protein were assayed for activities that cleave DEVD-AFC (left) and VEID-AFC (right), tetrapeptide substrates that correspond to preferred substrates of caspases 3 and 7 versus 6, respectively [61]. Results were compared to a standard curve of free AFC to calculate the product released per unit time. (B) After treatment with cytarabine (300 nM) or 17-allylamino17-demethoxygeldanamycin (17-AAG, 300 nM) as described [20], ML-1 human leukemia cells were simultaneously lysed and assayed for activity that cleaves DEVD-rhodamine 110. Error bars, range of duplicate samples from a single experiment. (C) Duplicates of the samples in (B) were stained with allophycocyanin-coupled annexin V to detect phosphatidylserine, a lipid that becomes exposed on the cell surface following caspase activation [62].

activity was also assayed in other subcellular fractions prepared from similar cell homogenates [18,22]. Recently developed kits that combine cell lysis and substrate addition in a single step provide a convenient alternative for many applications. In the example shown in Fig. 1B, aliquots that initially contained 2 · 104 cells before 48 h of drug treatment were treated with Apo-ONE Caspase-3/7 Reagent as described by the supplier. This proprietary reagent contains detergent to disrupt the plasma membrane and prepare a cell lysate for analysis of activity. Duplicate aliquots of cells were saved for DNA determination and annexin V binding assays. 2.4. Assays for caspase activity After preparation of cytosol by the classical method, aliquots containing 50 lg of protein were adjusted to 50 ll using ice cold buffer A containing 5 mM EDTA

and 1 mM DTT. Samples were diluted with 225 ll of freshly prepared 100 lM DEVD-AFC or VEID-AFC in buffer B, which consists of 25 mM HEPES (pH 7.5) and 0.1% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1propanesulfonate (CHAPS). Blanks contained 50 ll buffer A supplemented with EDTA and DTT + 225 ll 100 lM substrate in buffer B. Based on earlier studies showing that reactions were linear for at least 4 h [18], samples were incubated at 37 C for 2 h, diluted with 1.225 ml ice cold buffer B, and examined using a Sequoia-Turner spectrofluorimeter with excitation and emission wavelengths of 400 and 505 nm, respectively. Results were compared to a series of standards containing 0–1500 pmol of AFC in 50 ll buffer A (with 5 mM EDTA and 1 mM DTT) + 1.45 ml buffer B. After subtraction of the reagent blank, results in each sample were compared to the standard curve to derive the amount of AFC liberated per unit protein per unit time.

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Alternatively, in the case of the Apo-ONE homogenous caspase-3/7 kit, 100 ll aliquots of cells were mixed with 100 ll of working reagent and incubated for 120 min at 37 C. Fluorescence was then measured in a Molecular Devices SpectraMAX GeminiEM fluorescent plate reader using excitation and emission wavelengths of 485 and 530 nm, respectively. After determination of the amount of DNA in replicate aliquots of the cell lysates based on fluorescence observed after incubation of samples or standards with 50 lg/ml propidium iodide (excitation and emission wavelengths 536 and 617 nm, respectively) in 0.1% (w/v) sodium citrate containing 0.1% (w/v) Triton X-100, results were expressed as the relative amount of rhodamine 110 liberated per unit DNA per unit time. 2.5. Results obtained with these assays Typical results obtained with these assays are illustrated in Fig. 1. For example, treatment of DT40 cells with the topoisomerase II poison etoposide under conditions that produce apoptotic morphological changes in 40–60% of cells [23] results in DEVD-AFC and VEID-AFC cleavage activities of 250 pmol/min/mg protein and 70 pmol/min/ mg protein compared to negligible activities in the untreated cells (Fig. 1A). Deletion of the gene for caspase 6 has no effect on DEVD-AFC cleavage activity, consistent with previous claims that DEVD-AFC is a preferred substrate for caspases 3 and 7 [1]. As illustrated in Fig. 1B, an activity that cleaves DEVDrhodamine 110 increases after treatment of ML-1 cells with cytarabine and 17-AAG administered on various schedules. This activity is greatest when 17-AAG is administered in the continued presence of cytarabine, paralleling results obtained when apoptosis was quantified based on morphological changes or annexin V binding (Fig. 1C and Ref. [20]). 2.6. Trouble shooting the assays One difficulty often encountered in caspase assays is high (and variable) background of caspase activity in ‘‘control’’ cells. This can be diminished by removing the accumulated cells that have undergone spontaneous apoptosis before addition of the apoptotic stimulus, e.g., by removal of non-adherent cells from adherent cultures (by changing the medium) or by Ficoll-Hypaque sedimentation of leukemia cells before drug treatment. Other problems can include a small increase in activity and poor recovery of cytoplasmic protein because apoptosis is followed by rupture of the plasma membrane (so-called ‘‘secondary necrosis’’) with loss of cytoplasmic contents. This can be avoided by examination of earlier time points in the death process. 2.7. Strengths and limitations of the assays Each of the approaches outlined above has advantages and disadvantages. When samples harvested at multiple

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time points are subfractionated to resolve various organelles from one another, the classical fractionation approach outlined above can provide considerable temporal and spatial information about caspase activation. Moreover, this approach generates sufficient material to allow examination of multiple activities in replicate aliquots. For example, using replicate aliquots from one set of lysates, it is possible to assay for activities that cleave DEVD-AFC, VEID-AFC and LEHD-AFC, the preferred substrates of caspases 3 and 7, caspase 6 and caspase 9, respectively [1,24]. This approach does, however, require 108 cells per time point and is somewhat labor intensive. The single phase assay requires fewer cells and is more convenient if one only wants to document the appearance of caspase activity; but the single phase assay does not allow examination of caspase activity in different subcellular fractions because it disrupts cellular membranes. Moreover, a separate kit is required for each of the enzyme activities. In addition, both of these assays have important limitations. Because there is overlap in the ability of caspases to cleave various substrates [24,25], one cannot infer the caspase(s) that have been activated solely based on the substrates that are cleaved. For example, lysates prepared from cells lacking caspase 6 nonetheless contain a small amount of activity that is capable of cleaving VEID-AFC (Fig. 1A), illustrating the ability of other caspases, most notably caspase 3, to cleave this substrate. We have likewise demonstrated that caspase 3 will cleave the preferred caspase 9 substrate LEHD-AFC [26]. Conversely, DEVD-AFC, a preferred substrate for caspase 3, can also be cleaved by caspases 1, 2, 4, 6, 7, 8, 10, and 14 [25,27–32]. Thus, when DEVD-AFC cleavage is detected in a cell lysate, it is unclear whether this reflects caspase-3 activity or not. In fact, other classes of enzymes (e.g., the proteasome) can also cleave certain caspase substrates [33,34]. This potential for cleavage by other classes of proteases is not always reflected in the way commercial suppliers name their kits but must be taken into account when examining results. It is also important to emphasize that cells contain X chromosome-linked inhibitor of apoptosis (XIAP) protein, an endogenous inhibitor of caspases 3, 7, and 9 [35]. Depending upon the levels of XIAP, activity of these caspases can be underestimated or even undetectable in lysates from cells that exhibit other hallmarks of caspase activation [22]. For this reason it is important to perform complementary assays for caspase activation, particularly when caspase activity is low or undetectable. 2.8. Alternative approaches for detecting caspase activity Because peptide–AFC complexes are poorly cell permeable and free AFC leaks out of cells, the substrates described above are not useful for assessing caspase activity by fluorescence microscopy or flow cytometry. However, alternative substrates such as PhiPhiLux (OncoImmunin, Gaithersburg, MD) are suitable for these applications

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[36,37]. These types of substrates, which consist of a peptide containing a caspase cleavage site, a fluorescent moiety such as rhodamine 110 on one end, and a quencher on the other, generate a fluorescent signal when the fluorophore and quencher are separated upon peptide cleavage. Advantages of this approach include the ability to assess caspase activation on a single-cell basis. Disadvantages include the expense of these substrates, the need to load them into cells before applying an apoptotic stimulus (which can provide an additional stress for cells), and the fact that multiple proteases (including multiple caspases) could in principle cleave a particular substrate. Thus, even with this approach, complementary assays for caspase activation should be applied. 3. Analysis of caspase activation by immunoblotting 3.1. Overview Because activated caspases cleave intracellular polypeptides, it is possible to follow caspase activation by assessing the integrity of these substrates. Thus, immunoblotting remains an important approach for determining whether caspases have been activated. The availability of sera that specifically recognize caspase cleavage products (so-called ‘‘anti-neoepitope antibodies’’) has enhanced the usefulness of this approach. Because the zymogen forms of effector caspases are cleaved during their activation, it has also been suggested that blotting for these polypeptides can be utilized to determine whether caspases have been activated. On the other hand, the observation that initiator caspases are activated by dimerization (see above) makes immunoblotting for cleavage of initiator caspases less useful for assessing activation of these enzymes. 3.2. Reagents Rabbit polyclonal antibodies to procaspases 3 and 6 were from Cell Signaling Technology (Beverly, MA). Rabbit anti-protein kinase Cd and anti-ICAD were from Santa Cruz Biotechnology (Santa Cruz, CA) and Becton Dickinson Pharmingen (San Diego, CA), respectively. Monoclonal antibodies to poly(ADP)-ribose polymerase (PARP), topo I and procaspase 9 were kind gifts from Guy Poirier (Laval University, Ste.-Foy, Quebec), Y.-C. Cheng (Yale University, New Haven, CT) and Yuri Lazebnik (Cold Spring Harbor Laboratories, Cold Spring Harbor, NY), respectively. Antisera that recognize lamin A and cleaved caspase 3 were generated as previously described [38,39]. Vincristine was obtained from Sigma (St. Louis, MO). 3.3. Preparation of samples for SDS–PAGE In the example shown below, MDA-MB-468 cells were propagated in improved minimal essential medium (Invitrogen, Carlsbad, CA) containing 5% (v/v) heat-inactivated fetal bovine serum. When cells reached 20–30% confluence,

vincristine was added to a final concentration of 100 nM from a 1000-fold concentrated stock in DMSO. After a 72-h incubation, non-adherent cells were harvested and adherent cells were released by trypsinization. After recovery by sedimentation for 10 min at 100g, cells were washed twice with ice cold PBS and prepared for SDS–polyacrylamide gel electrophoresis as described [40]. Aliquots containing 50 lg of total cellular protein [assayed by the bicinchoninic acid method—Ref. [21]] were subjected to SDS–PAGE on gels containing linear 5–15% acrylamide gradients, transferred to nitrocellulose and probed with antibodies using standard techniques [41]. 3.4. Results obtained with these assays A typical result obtained using this approach is illustrated in Fig. 2. Treatment of MDA-MB-468 breast cancer cells with the spindle poison vincristine at the indicated concentration results in apoptotic morphological changes in 40–60% of the cells. Importantly, the apoptotic cells detach from the tissue culture flasks and are found exclusively in the floating fraction, whereas healthy cells adhere to the plastic. When whole cell lysates are subjected to SDS–PAGE followed by immunoblotting, a number of polypeptides, including the nuclear DNA repair protein PARP, the nuclear enzyme topoisomerase I, and lamin A are cleaved to characteristic fragments [1] that can be readily detected in the floating cells (Fig. 2A, lane 3). When the same blots are probed with antibodies that recognize procaspases, diminished levels of procaspases 3 and 6 are observed in the floating fraction (Fig. 2C, lane 3). Reaction with antiserum raised against IETD, the peptide that becomes the C-terminus of the active caspase 3 large subunit after cleavage of procaspase 3 by initiator caspases (Fig. 2B), indicates the presence of two species of cleaved caspase 3 exclusively in the non-adherent fraction (Fig. 2C, lane 3). The non-adherent fraction also contains diminished levels of procaspases 8 and 9 (Fig. 2C) and increased levels of cleaved caspases 8 and 9 (not shown, similar to cleaved caspase 3 data). 3.5. Strengths and limitations of the assays The commercial availability of high quality antibodies that recognize caspase cleavage products, including antineoepitope antibodies (Fig. 2B and C), has made it possible to detect procaspase cleavage during apoptosis. When detected, this cleavage can provide an important piece of evidence pointing to caspase activation. Several important limitations, however, make it imperative that cleavage of procaspases not be used as the sole criterion for caspase activation within cells. First, as indicated in the overview, initiator caspases appear to acquire at least some of their enzymatic activity without cleavage [7,42]. Thus, it is possible for caspases 8 and/or 9 to be active without altered mobility on immunoblots. Second, depending on the stability of caspase cleavage products, the level of cleaved cas-

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Fig. 2. Assessment of caspase activation by immunoblotting. (A) MDA-MB-468 human breast cancer cells were treated for 24 h with diluent (lane 1) or 100 nM vincristine (lanes 2 and 3), washed, and incubated for an additional 48 h in drug-free medium before adherent (A) or floating (F) cells were collected, washed, and lysed in 6 M guanidine hydrochloride [40]. Aliquots containing 50 lg of total cellular protein were subjected to SDS–PAGE, transferred to nitrocellulose, and incubated with antibodies that recognize the indicated caspase substrates [1]. Heat shock protein 90 (Hsp90) served as a loading control. (B) Diagrammatic representation of caspase 3 activation. Upstream caspases cleave between the large and small subunits, whereas removal of the prodomain is thought to involve the activity of caspase 3 itself [63,64]. X-ray crystallography has demonstrated that activation is accompanied by a conformational change that removes an occluding loop from the enzyme active site. Accordingly, it is possible to raise antibodies that specifically recognize activated caspases as a consequence the new appearance of a free C-terminal aspartate in the large subunit [39] (or new free Nterminal serine in the small subunit) or as a consequence of the conformational change [46]. The former are useful for immunoblotting (C) as well as immunohistochemistry (Fig. 3), whereas the latter are only useful for immunohistochemistry or immunoprecipitation. (C) Lysates from (A) were also probed with antibodies that recognize the indicated procaspases or cleaved caspase 3, which separates into two bands that differ by the presence (top band) versus absence (bottom band) of the prodomain [64]. The caspase resistant nucleolar protein B23 [65] served as a loading control.

pase might not accurately reflect the amount of caspase that has been activated. Third, the presence of potent caspase inhibitors within cells, notably XIAP, means that not all cleaved caspases 3, 7, or 9 molecules retain activity [22,43]. Accordingly, to determine whether caspases have been active within dying cells, it is important to assess whether other caspase substrates have been cleaved as well.

Because the list of caspase substrates now numbers >400 [6], it is relatively simple in most cases to blot for characteristic cleavage fragments of caspase substrates (Fig. 2A) in order to determine whether caspases have been activated. As with other methods, there are important limitations to the use of immunoblotting to assess caspase action. First, the method relies on the availability of high quality

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antibodies to the substrate and/or caspase being analyzed. It is difficult to assess the disappearance of a band at one molecular weight and appearance at a different molecular weight if the immunological reagent recognizes 10 bands on a blot. Second, because of instability of the caspase-generated fragments, some immunological reagents will recognize the full-length polypeptide but not the cleaved fragment (e.g., ICAD-L in Fig. 2A). Third, immunoblotting does not provide a quantitative assessment of the number of cells undergoing apoptosis. As illustrated in Fig. 2A, cleavage of PARP and lamin A can be incomplete even in a population in which >90% of cells exhibit apoptotic morphological changes. Using the extent of cleavage to assess the degree of apoptosis is even more problematic for antigens such as topo I, which are poor caspase substrates and consequently are cleaved later during apoptosis [44]. Finally, the cleavage of a particular substrate does not necessarily indicate which caspases have been activated. Lamin A, for example, is cleaved exclusively by caspase 6 in situ [19]. In contrast, PARP, which is classically viewed as a caspase 3 substrate, is reportedly cleaved by caspases 3, 7, 8, 9, and 10 in vitro and by caspases 7 and/or 9 in cells that lack caspase 3 [22,45]. 4. Detection of caspase activation by immunofluorescence or flow cytometry 4.1. Overview Because caspases undergo conformational changes upon activation [14], it should in principle be possible to generate immunological reagents that recognize only the active conformations of various caspases. Consistent with this view, antibodies that recognize the active conformation of caspase 3 have been described [46]. Because SDS–PAGE destroys polypeptide secondary and tertiary structure, these reagents are not useful for immunoblotting but can

be utilized for detecting active caspases by immunofluorescence or flow cytometry. In addition, some of the anti-neoepitope antibodies that recognize C- or N-terminal amino acids liberated during caspase cleavage are useful for immunofluorescence or flow cytometry-based methods. 4.2. Materials and methods MDA-MB-468 human breast cancer cells were treated for 24 h with 0.1% (v/v) DMSO lacking or containing 100 nM paclitaxel, washed, and incubated in drug-free medium for an additional 24 h. Adherent and non-adherent cells were sedimented onto glass slides at 60g for 5 min in a cytocentrifuge, air dried, fixed in 3% (w/v) paraformaldehyde in PBS, permeabilized with TBST buffer [150 mM NaCl, 50 mM Tris–HCl (pH 7.4 at 21 C), 0.1% (w/v) Tween 20], and treated with 5% fetal bovine serum in TBST to block nonspecific binding sites. Cells were then stained with affinity-purified antibodies that recognize IETD (Cell Signaling Technology), the sequence that becomes the C-terminus of the caspase 3 large subunit when procaspase 3 is cleaved by initiator caspases (Fig. 2B), followed by fluorescein-labeled anti-rabbit IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD) according to the instructions of the supplier. After the last wash, samples were stained with 1 lg/ml Hoechst 33258, mounted in Vectashield (Vector Laboratories, Burlingame, CA) and examined using a Zeiss LSM510 confocal microscope. 4.3. Results obtained with this method Results obtained when MDA-MB-468 cells are stained with antibodies that specifically recognize cleaved caspase 3 are illustrated in Fig. 3. Cleaved caspase 3 is detected in both the cytoplasm and fragmented nuclei (arrowheads) of apoptotic cells but not in nearby non-apoptotic cells

Fig. 3. Detection of cleaved caspase 3 by immunofluorescence. MDA-MB-468 cells harvested 48 h after initiation of treatment with 50 nM paclitaxel [66] were sedimented onto coverslips using a cytocentrifuge, fixed in paraformaldehyde, and stained with antibodies that recognize free aspartate at the Cterminus of the caspase 3 large subunit after cleavage of the sequence IETDflS in procaspase 3 (see Fig. 2B and C). In apoptotic cells, cleaved caspase 3 was not only detected in the cytoplasm, but also in some of the condensed nuclear fragments (arrowheads). The cleaved caspase was not detectable in the nuclei or cytoplasm of cells with a normal chromatin pattern (asterisks).

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whose nuclei show a distribution of chromatin characteristic of healthy cells (asterisks). Complementary biochemical experiments have demonstrated that the caspase substrates PARP, topoisomerase I, ICAD-L, b-catenin, gelsolin, and protein kinase Cd as well as procaspases 3, 6, 7, 8, 9, and 10 (but not 2) are cleaved in these cells [22]. 4.4. Strengths and limitations of this approach Published results have demonstrated that these types of reagents can also be used to assess caspase cleavage by flow cytometry [47] and enzyme linked immunosorbent assay [48]. With both immunofluorescence and flow cytometry, the strength of the approach is that it can be used to obtain quantitative data about the percentage of cells staining above a particular threshold. The specificity of some of the reagents that recognize conformational epitopes or cleaved caspases is sufficiently high that one can also say with near certainty whether a particular caspase has assumed an active conformation or has been cleaved, respectively. Although the number and quality of available reagents continues to improve, these assays have been limited in part by the lack of availability of conformation-sensitive antibodies to most of the active caspases and potential cross-reactivity of anti-caspase neoepitope antibodies with other polypeptides that are cleaved during apoptosis to render similar sequences accessible for binding.

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5.2. Materials Biotin-X-VAD(OMe)-fmk and zEK(bio)D-aomk were obtained from Calbiochem and the Osaka Peptide Institute (Osaka, Japan), respectively. Synthesis of derivatives of zEK(bio)D-aomk with longer linker arms and zEK(DNP)D-aomk was recently described [52]. Peroxidase-coupled streptavidin was from Amersham Biosciences. Monoclonal anti-DNP was from Molecular Probes. 5.3. Preparation of subcellular fractions Because of poor cell penetration, many of the affinity labels are employed to tag active caspases after subcellular fractionation. Cytosol and other subcellular fractions can be prepared as previously described (Section 2.3 and Refs. [18,22]). Alternatively, in the example shown in Fig. 4B, DT40 cells treated with diluent or 10 lM etoposide for 5 h were sedimented at 300g for 5 min, resuspended in 5 ml ice cold buffer C [50 mM PIPES (pH 7.0), 50 mM KCl, 10 mM EGTA, 2 mM MgCl2] and sedimented again. All further steps were performed at 4 C. The cells were

5. Affinity labeling 5.1. Overview Another approach for the detection of active caspases involves the covalent modification of the caspase active site with a substrate-like molecule that contains both an inhibitory group and a reporter moiety. Covalent binding of such an affinity label facilitates subsequent detection of the activated caspase by immunoblotting, fluorescence microscopy or flow cytometry depending upon the reporter group. A number of variations on the basic molecular structure of the affinity label have been examined. Biotinylated tri- or tetrapeptides linked to fluoromethyl ketone (fmk), a group that covalently inhibits sulfhydryl proteases [49], have been extensively used in these studies. Based on diminished reactivity and the resulting diminished propensity for nonspecific binding, acyloxymethylketones (aomks) have been preferred by some investigators [49,50]. The reporter moiety has been attached either to the amino acid immediately upstream of the scissile aspartate (the P2 amino acid), which is solvent exposed and therefore able to accommodate a bulky substituent when the inhibitor is bound to the enzyme [51], or to the N-terminus of the peptide. Reporter groups have included biotin, which can be detected with labeled streptavidin; fluorescein, which renders the labeled protein fluorescent; or 2,4-dinitrophenol (DNP), which reacts with antibodies.

Fig. 4. Labeling of active caspases by acyloxymethylketones. (A) MDAMB-468 cells harvested 48 h after initiation of treatment with diluent ( ) or 50 nM paclitaxel (T) were fractionated into cytosol (C) and nuclei (N) as previously described [66]. Aliquots containing 40 lg of total protein were reacted with zEK(bio)D-aomk, subjected to SDS–PAGE and transferred to nitrocellulose. Bound affinity label was visualized using peroxidase-coupled streptavidin and enhanced chemiluminescence reagents. Cytosol from apoptotic HL-60 cells (lane 1) served as a positive control. Previous two-dimensional gel electrophoresis [18] demonstrated that the 17–22 kDa bands correspond to cleaved forms of caspases 3 and 6. Dotted lines indicate removal of intervening lanes. Note the binding of streptavidin to a series of polypeptides in the absence of caspase activation (lane 2 and particularly lane 4). These appear to represent endogenous biotinylated polypeptides. (B) cytosol from control (lane 1) or etoposidetreated DT40 cells (lane 2) was reacted with the dinitrophenol (DNP)containing affinity label zEK(DNP)D-aomk [52], subjected to SDS– PAGE, and blotted with anti-DNP antiserum. Numbers at left, molecular weights of marker proteins in kDa.

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resuspended in 1 ml buffer D (buffer C supplemented with 0.1 mM PMSF, 20 lM cytochalasin B, 1 mM Na3VO4, and 1 lg/ml of chymostatin, pepstatin, leupeptin, and antipain), transferred to a 1.5 ml microfuge tube, sedimented at 800g, resuspended in a volume of buffer D equal to the cell pellet, and lysed by three cycles of freezing in liquid nitrogen alternating with thawing. After sedimentation for 1 h at 157,000gmax using a Beckman TLA100.1 rotor, aliquots were removed for protein determination. Samples were then diluted to 5 mg protein/ml in buffer D and frozen at 80 C until affinity labeling and analysis was performed. 5.4. Affinity labeling To perform affinity labeling, 40 lg of cytosolic protein was incubated with 5 lM zEK(bio)D-aomk or 2 lM zEK(DNP)D-aomk (added from a 100-fold concentrated stock in DMSO) for 30 min at 30 C. At the conclusion of the incubation, samples were diluted with 1/2 volume of 3-fold concentrated SDS-sample buffer [45% (w/v) sucrose, 9% (w/v) SDS, 6 mM EDTA and 10% (v/v) bmercaptoethanol in 150 mM Tris–HCl, pH 6.8], boiled for 5 min and subjected to SDS–PAGE on a 16% (w/v) acrylamide gel. After transfer to polyvinylidene fluoride membrane, the blot was blocked in solution containing 5–10% (w/v) powdered nonfat milk. Polypeptides labeled with zEK(bio)D-aomk were then detected by incubating the blot with peroxidase-coupled streptavidin for 3–4 h followed by washing and exposure to enhanced chemiluminescence reagents as typically performed with a peroxidase-coupled secondary antibody. Alternatively, polypeptides labeled with zEK(DNP)D-aomk were detected by sequential reaction with murine anti-DNP monoclonal antibody and peroxidase-coupled anti-mouse IgG essentially as described for immunoblotting. 5.5. Results obtained with this method Typical results obtained with this method are illustrated in Fig. 4. Treatment with an apoptotic stimulus results in labeling of a series of bands at 16–20 kDa. Additional experiments have shown that these bands correspond to the large subunits of cleaved caspases 3 and 6 [18,53]. Labeling of these bands is diminished by pretreatment of the cell extracts with the irreversible caspase inhibitor DEVD-fmk [26]. In contrast, high molecular weight bands that presumably correspond to endogenous biotinylated polypeptides (Fig. 4A) continue to bind streptavidin after the DEVD-fmk pretreatment [26]. Labeling of these high molecular weight bands is not observed when zEK(DNP)D-aomk and anti-DNP antibodies are utilized to detect active caspases (Fig. 4B). 5.6. Emerging variations on this method Although this technique was originally designed to allow detection and identification of active caspases at a time

when antibodies were not available [18,54], several observations have sustained interest in this approach. It has been observed that prolonged incubation of cells with Biotin-XVAD(OMe)-fmk, a derivative of the broad spectrum caspase inhibitor zVAD-fmk [55], before application of an apoptotic stimulus loads enough affinity label into cells to allow caspase derivatization as these proteases are activated [16]. If lysates from these cells are then denatured (see below), purified on streptavidin-coupled agarose, and subjected to immunoblotting with anti-caspase antibodies, caspases that are activated at the apex of a proteolytic cascade can be identified [16]. Alternatively, after affinity purification, caspases can be assayed for posttranslational modifications, e.g., by purification from cells that have been labeled with 32P-orthophosphate followed by autoradiography [56] or by mass spectrometry [53]. Although the application of these approaches had been limited by relatively poor penetration of affinity label into cells, the recent development of more cell permeable DNP-coupled inhibitors promises to eliminate this problem [52]. Moreover, the use of longer linker arms between the peptide and reporter group circumvents the need for denaturation before affinity purification [52], permitting the search for polypeptides that might be bound to active caspases. Finally, it has been reported that fluorochrome-labeled affinity ligands can penetrate into cells and become covalently bound to activated caspases, thereby allowing detection of these enzymes by fluorescence microscopy or flow cytometry [57,58]. 5.7. Strengths and limitations of this approach The affinity labeling methods outlined above are uniquely capable of permitting identification of the earliest caspases activated in cells [16] or the affinity purification of caspases so that binding partners or posttranslational modifications can be studied [52,53,56]. In addition, because these techniques rely upon recognition of substrate-like peptides, they can be applied to species for which anti-caspase antibodies do not exist. Despite their potential usefulness, however, all of the methods that employ affinity labels also have some important limitations. First, these techniques require reagents that are expensive and might require a difficult custom synthesis. Second, endogenous biotinylated polypeptides will be detected or co-purified when biotin is used as the reporter group (Fig. 4A). Third, it is important to recognize that reactive groups utilized to derivatize the caspase active site are attacked relatively nonspecifically by sulfhydryl groups and other nucleophiles [49]. Thus, it is not surprising that zVAD-fmk and AcDEVD-fmk can irreversibly inhibit cathepsins B and H [32,59] or that Biotin-X-VAD-fmk can affinity label cathepsin B [59]. This ability to derivatize other enzymes particularly limits the usefulness of fluorochrome-coupled affinity ligands as the sole means of detecting caspase activation [60]. Conversely, it is also important to recognize that not all of the caspases react with each of the affinity

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