Comparison of the C2A Domain of Synaptotagmin-I and Annexin-V As

Apr 19, 2010 - We show here that the C2A domain of Synaptotagmin-I, which had been .... chromatography (see Methods 1 in Supporting Information). The...
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Bioconjugate Chem. 2010, 21, 884–891

Comparison of the C2A Domain of Synaptotagmin-I and Annexin-V As Probes for Detecting Cell Death Israt S. Alam,†,§ Andre A. Neves,†,§ Timothy H. Witney,‡ Joan Boren,§ and Kevin M. Brindle*,‡,§ Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom, and Cancer Research UK Cambridge Research Institute, Li-Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, United Kingdom. Received October 8, 2009; Revised Manuscript Received March 9, 2010

The induction of apoptosis is frequently accompanied by the exposure of phosphatidylserine (PS) on the cell surface, which has been detected using radionuclide and fluorescently labeled derivatives of the PS-binding protein, Annexin V. The fluorescently labeled protein has been used extensively in vitro as a diagnostic reagent for detecting cell death, and radionuclide-labeled derivatives have undergone clinical trials for detecting tumor cell death in vivo following treatment. We show here that the C2A domain of Synaptotagmin-I, which had been fluorescently labeled at a single cysteine residue introduced by site-directed mutagenesis, detected the same levels of cell death as a similarly labeled Annexin-V derivative, in drug-treated murine lymphoma and human breast cancer cell lines in vitro. However, the C2A derivative showed significantly less binding to viable cells and, as a consequence, up to 4-fold more specific binding to apoptotic and necrotic cells when compared with Annexin-V. C2A offers a potential route for the development of a new generation of more specific imaging probes for the detection of tumor cell death in the clinic.

INTRODUCTION The detection of cell death in the clinic is of considerable interest for assessing the effectiveness of therapy and for determining the progression of disease (1, 2). In oncology, the induction of tumor cell death following treatment can be a good predictive indicator of treatment outcome, a positive correlation having been observed, for example, between the levels of tumor cell death post treatment and patient survival rates, in human breast cancer (3, 4). Apoptosis is frequently associated with the externalization of negatively charged phospholipids, such as phosphatidylserine (PS), on the outer leaflet of the plasma membrane bilayer, where they direct engulfment of the apoptotic cell by neighboring phagocytes (5). PS is also exposed during subsequent necrosis, due to disruption of plasma membrane integrity. The detection of PS exposure by Annexin V (AnxV) has often been used to detect apoptosis. AnxV is a 36 kDa protein that binds with high affinity and in a Ca2+-dependent manner to the PS exposed on dying cells. Radionuclide derivatives of AnxV reached early-stage clinical trials and were shown to detect cell death in patients with myocardial infarcts (6), lymphoma, and lung cancer (7, 8). There were, however, problems with biodistribution (9, 10). The C2A domain of Synaptotagmin I is a 14.7 kDa protein, which like AnxV binds to PS in a Ca2+-dependent manner (11, 12). A glutathione-S-transferase (GST)-tagged version of C2A was shown to detect cell death in vitro and in vivo using radionuclide and MR imaging techniques (13-15). The protein is of interest since, due to its smaller size, it may show better penetration of tumor tissue than AnxV and also better clearance of unbound material, thus generating better tissue contrast. * Corresponding author. Kevin M. Brindle, Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK., Tel: +44 (0)1223 333674, Fax: +44 (0)1223 766002, Email: [email protected]. † Equal contributions. § Cancer Research UK Cambridge Research Institute. ‡ University of Cambridge.

In previous studies with the C2A domain of synaptotagmin, an MRI contrast agent was attached to the ε-amino group of surface lysine residues (13, 15, 16). There are 14 lysine residues in C2A and a total of 35 in the C2A-GST fusion protein (15). Attachment of labels to multiple lysine residues inevitably results in a reagent that consists of a heterogeneous mixture of molecular species. Moreover, in the case of an MRI agent based on C2A-GST, modification of these lysine residues with MRdetectable Gd3+-chelates was shown to lower the affinity of the protein for phosphatidylserine (PS) (15). We describe here a site-directed mutant of C2A, C2Am, in which we have replaced a serine residue that is distant from the PS-binding site, with a single cysteine residue (S78C). Since there are no other cysteine residues in the protein, this provides a unique site for the modification of the protein with fluorescent, radionuclide, or MR-detectable labels. In this study, we have assessed the potential of a fluorescent derivative of C2Am to detect cell death in murine lymphoma and human breast cancer cell lines, by comparing both its sensitivity and specificity for detection of apoptotic and necrotic cells with that of a fluorescent derivative of AnxV that is available commercially.

EXPERIMENTAL PROCEDURES Materials. All tissue culture reagents, SYTOX Green dead cell stain, and Annexin V-Alexa Fluor647 (AnxV-AlxF647) were from Invitrogen (Paisley, Renfrewshire, UK). The green Polycaspases FLICA kit was from Immunochemistry Technologies LLC (Bloomington, MN, USA). All chemicals were of analytical grade from Sigma-Aldrich Co. Ltd. (Poole, Dorset, UK), unless stated otherwise. Etoposide (EPOSIN) was from PCH Pharmachemie, (Haarlem, Netherlands). Murine lymphoma (EL-4) cells were obtained from the American Type Culture Collection (Teddington, Middlesex, UK) and MDA-MB-231 cells were from European Collection of Cell Cultures (Salisbury, Wiltshire, UK). Construction, Overexpression, and Purification of C2A(S78C). The Rattus norVegicus synaptotagmin I sequence was obtained from the NCBI Genbank service (sequence NM_001033680).

10.1021/bc9004415  2010 American Chemical Society Published on Web 04/19/2010

Detection of Cell Death Using C2A vs Annexin-V

Appropriate primers for mutagenesis were designed using Quikchange Primer Design software. The C2A-containing pGEX-2T vector (GE Healthcare, Chalfont St Giles, UK) was isolated from E. coli BL21 cells and mutagenesis performed using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA). E. coli DH5a competent cells were transformed with the mutant plasmid by heat shock and the amplified vector then isolated and sequenced. Finally, E. coli BL21 cells were transformed with the vector by electroporation. The proteins were expressed as 42-kDa GST-tagged fusion proteins, following induction using isopropyl-β-D-thiogalactoside (IPTG), and the S78C mutant was purified using affinity chromatography (see Methods 1 in Supporting Information). The extent of purification was analyzed using SDS-PAGE. Protein concentrations were determined using a Non-Interfering Protein AssayTM kit (NIPA from Merck Chemicals Ltd., Nottingham, UK) and by densitometry for AnxV-AlxF647 (see Methods 2 in Supporting Information). Production of Fluorescently Labeled Protein. The mutant protein (C2Am) was covalently labeled with the fluorophore Alexa Fluor 647 (AF647), by reaction of the cysteine-78 residue with the maleimide group of Alexa Fluor 647 C2 maleimide (Invitrogen). Briefly, the protein was reduced using 10 mM DTT for half an hour at room temperature. The protein was then washed in a 5 kDa vivaspin concentrator (Sartorius, Epsom, UK) in HNE buffer (20 mM HEPES, 100 mM NaCl, 5 mM EDTA, pH 7.4). The protein was kept at a concentration in the range 50-100 µM, and an approximately 10-fold molar excess of the fluorescent maleimide dye was added. The reaction was allowed to proceed on a magnetic stirrer at 4 °C overnight. The modified protein was separated from unreacted dye by gel filtration using a Hi Load Superdex 75 26/60 prep grade column (GE Healthcare). Electrospray ionization (ESI) mass spectrometry was used to confirm modification of the protein, which showed a single species of approximately 17 204 Da (C2AmAF647) (see Methods 3 and Figure S1 in Supporting Information). Surface Plasmon Resonance (SPR). Protein affinity of C2Am and C2Am-AF647 for PS-containing liposomes was estimated using the Biacore T100 (Biacore, GE Healthcare). (See Methods 4 in Supporting Information). Cell Culture. EL4 murine lymphoma and MDA-MB-231 human breast cancer cells were propagated in RPMI 1640 media, supplemented with 10% fetal calf serum and 2 mM L-glutamine. Cell number and viability were monitored using Trypan blue dye staining. EL4 cell death was induced by addition of 5 µM etoposide for up to 24 h and MDA cell death by treatment with doxorubicin (1 µg/mL) for up to 96 h. Detection of Cell Death Using Flow Cytometry. Cell pellets (106 cells) were washed in ice-cold HEPES-buffered saline (10 mM HEPES, 150 mM NaCl, 2 mM CaCl2, pH 7.4) with 1% fetal calf serum and resuspended in 100 µL of the same buffer containing C2Am-AF647, which was used at concentrations between 50 nM to 1.0 µM, or AnnexinV-AF647 (Invitrogen), which was used at concentrations between 1 and 20 nM, in combination with SYTOX Green (Invitrogen; 50 nM), and incubated for 15 min at 37 °C. The resulting mixture was washed twice, kept briefly on ice, and then analyzed in an LSRII cytometer (BD Biosciences, Rockville, MD USA), with 20 000 cells counted per event. For detection of active caspases, EL4 or MDA cells were washed and then incubated with Poly Caspases FLICA for 30 min at 37 °C (300-fold diluted stock). A further wash step was performed before incubation with C2Am and AnxV. The same washing steps were applied as in all the other flow cytometry studies. All experiments were performed at least three times.

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Image Analysis of C2Am and AnxV Binding to Dying Cells. EL4 cell pellets (5 × 106 cells) were washed in ice-cold HEPES-buffered saline. The pellets were resuspended in 1 mL of warm buffer containing the mitochondrial stain MitoTracker Orange CMTMRos (Invitrogen, 100 nM) and incubated for 30 min at 37 °C. Cells were washed once and resuspended in icecold HEPES-buffered saline with 3% BSA for 30 min at 4 °C. Cells were then resuspended in 100 µL HEPES-buffered saline containing either C2Am-AF647 or AnxV-AF647 (Invitrogen), which were used at concentrations of 200 nM and 4 nM, respectively, and incubated for 15 min at 37 °C. The resulting mixture was then washed twice and resuspended in 100 µL icecold buffer containing the nucleic acid stain, 7-amino-actinomycin D (7-AAD) (Invitrogen, 20 µg/mL) and kept on ice briefly before samples were loaded into an ImageStream system (Amnis Corporation, Seattle, WA, USA), and a file of 5000 cells was collected in the standard collection mode. Data files were analyzed using IDEAS software (Amnis, Seattle, WA) to obtain fluorescence images of individual cells. Statistical Analysis. Two-way ANOVA and subsequent Mann-Whitney’s test were used for all analyses. P-values below 0.05 were considered to be statistically significant.

RESULTS Preparation of C2A(S78C). The serine residue at position 78 of the C2A domain of Synaptotagmin-I (position 217 of Synaptotagmin-I), was replaced with a cysteine residue, using site-directed mutagenesis. Three candidate amino acid residues were selected for replacement: Ser217, Gln154, Asn248. SDS PAGE of the three GST-C2A mutants (S217C, N248C, and Q154C) was used to assess the levels of expression before and after IPTG induction. All vector-transformed bacteria expressed the mutant proteins to some degree, but a much higher level of overexpression was obtained for S217C. The isolated C2A domain [C2A(S78C)] was prepared as described in Experimental Procedures. Synthesis and Characterization of C2A(S78C)-Alexa Fluor 647. C2A(S78C) was reacted with a maleimide derivative of Alexa Fluor 647. The modified protein (C2Am), gave a single peak on electrospray ionization mass spectrometry, with the expected mass of 17 204 Da (see Figure S1 in Supporting Information). SDS-PAGE also showed a single band of the expected mass (see Figure S2 in Supporting Information). Surface plasmon resonance (SPR) measurements gave similar dissociation constants (Kd) for the unmodified and modified proteins for PS of 55.4 ( 3.5 nM and 71.0 ( 6.9 nM, respectively. The dissociation constant of wild-type C2A for PS is approximately 20 nM (16). These dissociation constants were corroborated by kinetic analysis of the SPR data (see Table ST1 in Supporting Information). The kon values determined from the kinetic analysis for the unmodified and modified proteins were 2.01 × 106 M-1 s-1 and 2.14 × 106 M-1 s-1, respectively, and the koff values were 0.110 s-1 and 0.188 s-1, respectively. Analysis of Fluorescently Labeled Annexin V. A commercial preparation of Annexin V (AnxV), that had been labeled with the same fluorophore (Alexa Fluor 647, or AF647) as C2Am, was analyzed by SDS-PAGE, which showed bands at ca. 35 kDa and ca. 65 kDa, which were thought to be AnxV and BSA, respectively (see Figure S3a in Supporting Information). The latter is an additive in the preparation, at 0.1% (w/ v). Fluorescent imaging of the same gel showed that the band at ca. 35 kDa was fluorescent (647 nm excitation), confirming it as AF647-labeled AnxV (see Figure S3b in Supporting Information). The intensity of this band indicated that it was approximately 2% (w/w) of the total protein concentration (determined to be 800 µg/mL), and therefore, the concentration of AnxV was estimated to be approximately 460 nM in this commercial preparation of the protein.

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Figure 1. Images of EL4 cells at different stages of cell death. Bright field images (A); fluorescent images of cells stained with MitoTracker orange (B); fluorescent images of cells stained with the DNA stain, 7AAD (7-amino-actinomycin) (C); fluorescent images of cells stained with AnxV-AF647 or C2Am-AF647 (D); overlays of images shown in A and D (E); overlays of images shown in C and D (F). C2Am was used at a concentration of 200 nM and AnxV at 4 nM.

Flow Cytometry of Dying Cells Stained with C2Am or AnxV. Images were acquired from etoposide-treated EL4 cells that had been sorted according to the level of binding of C2Am or AnxV. Viable cells, which did not stain with C2Am or AnxV (Figure 1d), had a rounded morphology and an intact plasma membrane (Figure 1a), showed mitochondrial activity (Figure 1b) and no nucleic acid staining (Figure 1c). In contrast, early apoptotic cells that stained with C2Am or AnxV (Figure 1d)

Alam et al.

appeared irregular, with blebbing of the plasma membrane (Figure 1a), but still showed mitochondrial activity (Figure 1b) and no nucleic acid staining (Figure 1c). Late apoptotic/necrotic cells showed no detectable mitochondrial activity (Figure 1b), pronounced nucleic acid staining (Figure 1c), which demonstrated loss of plasma membrane integrity, and C2Am or AnxV staining (Figure 1d). Overlays of C2Am or AnxV staining with the bright field images are shown in Figure 1e, demonstrating that both probes stain the EL4 cell membrane of early apoptotic cells. Overlays of nucleic acid staining with the corresponding images of C2Am and AnxV staining are shown in Figure 1f, demonstrating that both C2Am and AnxV accumulate intracellularly in late apoptotic/necrotic EL4 cells. Detection of Cell Death Using Flow Cytometry and C2Am or AnxV. The facility of C2Am to detect cell death by flow cytometry in drug-treated murine lymphoma (EL4) and human breast cancer (MDA-MB-231, hereafter referred to as MDA) cell lines was compared with detection using AnxV. Cell death was induced by treatment of EL4 and MDA cells with etoposide or doxorubicin, respectively (Figures 2 and 3). Treatment of cells with DNA damaging agents, such as etoposide and doxorubicin, results in activation of poly ADPribose polymerase (PARP) and, since NAD+ is a substrate for PARP, depletion of the cellular NAD(H) pool (17). As a consequence, NADH autofluorescence is decreased considerably upon the induction of cell death using DNA damaging agents, and this decrease occurs relatively early after etoposide treatment in EL4 cells (18). Therefore, we identified apoptotic cells by their low NADH autofluorescence (green symbols), necrotic cells by their low NADH autofluorescence and high SYTOX green fluorescence (SYTOX green is a marker of cell necrosis) (orange symbols), and viable cells by their high NADH autofluorescence and low SYTOX green fluorescence (blue symbols) (Figure 3a,b and i,j). In this study, we have not tried to distinguish between primary necrosis, that is cells that have become necrotic without engaging the apoptotic pathway, and secondary necrosis, in which late apoptotic cells have become necrotic (19). Instead, when we refer to late apoptotic or necrotic cells, we are referring to cells with compromised plasma

Figure 2. Binding of C2Am and AnxV to viable, apoptotic, and necrotic EL4 and MDA cell populations. Cell death was induced in EL4 and MDA cells by treatment with etoposide or doxorubicin, respectively, for the specified times. The fractions of apoptotic (4), necrotic (0), and viable cells (1) were assessed by flow cytometric measurements of NADH autofluorescence (Exc, λ ) 350; Em, λ ) 475 nm) and Sytox green staining (Exc, λ ) 504; Em, λ ) 523 nm) (a and e). Binding of fluorescently labeled derivatives of C2Am (O) and AnxV (9) to the viable (b,f), apoptotic (c,g), and necrotic (d,h) cell populations was also assessed by flow cytometry, where binding is expressed as the mean fluorescence intensity (the total fluorescent signal for this population was divided by the number of cells and is expressed in arbitrary units). The fluorescence intensities used to calculate the MFIs were standardized to autofluorescence levels in the Alexa Fluor 647 channel. Data shown are means ( SEM (n ) 3; error bars lie within the symbols when not visible) for each time point. C2Am was used at a concentration of 200 nM and AnxV at a concentration of 4 nM. Differences were significant at the following: *P < 0.05, plot a-6 and 12 h, b-2 h, c-24 h; **P < 0.01, a-8 h, b-4 and 14 h, d-48 h; ***P < 0.001, a-10 and 14 to 24 h, b-0 and 12 h, c-48 to 96 h, d-0, 24, 72, and 96 h.

Detection of Cell Death Using C2A vs Annexin-V

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Figure 3. Specificity of C2Am and AnxV binding to viable, apoptotic, and necrotic EL4 and MDA cell populations. Cell death was induced by treatment of EL4 and MDA cells with etoposide or doxorubicin, respectively, for the specified times. The fractions of apoptotic (green symbols), necrotic (orange symbols), and viable cells (blue symbols) were assessed by flow cytometric measurements of NADH autofluorescence (Exc, λ ) 350; Em, λ ) 475 nm, x-axis) and Sytox green nuclear staining (Exc, λ ) 504; Em, λ ) 523 nm, y-axis) (a, b, i, and j). Binding of Alexa Fluor 647-labeled C2Am or AnxV (Exc, λ ) 647; Em, λ ) 670 nm, x-axis) to these cell populations is shown in c and d and k and l for C2Am and in e and f and m and n for AnxV. The specificity of binding was assessed by plotting the mean fluorescence intensity (MFI) ratios for the Alexa Fluor 647 following binding of C2Am (O) and AnxV (9) to the indicated cell populations, where these cell populations were identified by their NADH autofluorescence and Sytox green nuclear staining (g,h and o,p). The fluorescence intensities used to calculate the MFIs were standardized to autofluorescence levels in the Alexa Fluor 647 channel. Data shown are means ( SEM (n ) 3, error bars lie within the symbols when not visible) for each time point. Differences were significant at the following: *P < 0.05, plot s-48 h; **P < 0.01, h-12 h, i-14 h, q-24 and 72 h, r-24 h; ***P < 0.001, g-0 to 24 h, h-0 to 10 and 14 h, i-0 to 12 h, q-0 and 48 h, r-72 and 96 h, s-72 and 96 h. Sytox SYTOX green; AF647 ) Alexa Fluor 647. Table 1. Correlation Factors (R2) for Best Fit of EL4 or MDA Cell Populations (% Cells) Identified by C2Am or AnxV and by Measurements of NADH UV Autofluorescence and SYTOX Green Staining viable

apoptotic

necrotic

C2Am vs AnxV EL4 MDA

0.9641 0.9947

0.9333 0.9913

0.9919 0.9936

C2Am vs NADH and SYTOX Green EL4 MDA

0.9983 0.9734

0.9859 0.9965

0.9969 0.9840

AnxV vs NADH and SYTOX Green EL4 MDA

0.9975 0.9677

0.9568 0.9913

0.9986 0.9936

membrane integrity and which therefore stain with the nucleic acid stains SYTOX green or 7-AAD. Both C2Am (Figure 3c,d and k,l) and AnxV (Figure 3e,f and m,n) bound to the apoptotic and necrotic cell fractions identified by measurements of NADH autofluorescence and SYTOX green staining. Moreover, the cell populations identified by either protein were well-correlated with each other and with the cell populations identified by measurements of NADH autofluorescence and SYTOX green staining (Table 1). However, C2Am showed better specificity for detecting apoptotic and necrotic cells, when compared with AnxV, which can be explained by the greater binding of AnxV to viable cells. Viable EL4 and MDA cells (Figure 2b and f, respectively) showed consistently higher absolute staining with AnxV (up to 4-fold, P < 0.001,

for EL4 cells and up to 2-fold, P < 0.01, for MDA cells) when compared with C2Am, despite being used at a 50-fold lower concentration (4 nM AnxV and 200 nM C2Am). Although apoptotic and necrotic EL4 and MDA cells generally showed more labeling with AnxV than C2Am (Figure 2c,d and g,h), the effects of this on the specificity of detection of apoptotic and necrotic cells was more than offset by the greater binding of AnxV to the viable cells. This improved specificity can readily be appreciated from plots of the ratios of the mean fluorescence intensities (apoptotic and necrotic versus viable) following binding of C2Am and AnxV. The ratios of the mean fluorescence intensities (MFI) for apoptotic/viable (Figure 3g and o) and necrotic/viable (Figures 3 h and p) cells were higher for C2Am than for AnxV for the majority of the time following drug treatment, for both EL4 and MDA cells. C2Am showed greater specificity for apoptotic cells (P < 0.05 for EL4 and MDA cells) and necrotic cells (P < 0.05 for EL4 cells and P < 0.01 for MDA cells) when compared to AnxV. Apoptotic and necrotic MDA cells (Figure 3i and j, represented by the green and orange symbols) showed similar levels of SYTOX green staining to viable cells (in blue). Compare Figure 3a and b for EL4 cells with Figure 3i and j for MDA cells. However, doxorubicin is fluorescent and its emission overlaps with that of SYTOX Green (20), and therefore, this effect was probably due to the presence of the drug in the viable MDA cells in this particular experiment. Comparison of Cell Labeling with C2A and AnxV with Caspase Activation. As a further independent measure of cell death, we assessed caspase activation using a Poly Caspases

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Figure 4. Dual scatter plots of active caspase labeling (Poly Caspases FLICA carboxyfluorescein; Exc, λ ) 490; Em, λ ) 530 nm, y-axis) versus labeling with C2Am (b and g) or AnxV (c and h) (Alexa Fluor 647; Exc, λ ) 647; Em, λ ) 670 nm, x-axis) of EL4 cells treated with etoposide (5 µM) for 16 h (a-c) and MDA cells treated with doxorubicin (1 µg/mL) for 72 h (f-h). Apoptotic (green), late apoptotic/necrotic (orange), and viable (blue) EL4 or MDA cells were identified based on their levels of NADH autofluorescence and staining with SYTOX green (a, EL4 cells; f, MDA cells). Mean fluorescence intensities (MFI) following FLICA-labeling of the different EL4 and MDA cell populations is shown in (d) and (i), respectively. Data shown are means ( SEM (n ) 3) (***P < 0.001). C2Am and AnxV labeling (Alexa Fluor 647; Exc, λ ) 647; Em, λ ) 670 nm, x-axis) of the 5% of the viable cell population (purple in b and c and g and h) that showed the lowest FLICA labeling are shown for EL4 (e) and MDA (j) cells against the frequency of events. Viable EL4 and MDA cells stained more with AnxV (black line) than C2Am (shaded gray area) (P < 0.05, for EL4 and P < 0.001, for MDA). MFI values, in arbitrary units (a.u.), were standardized to green autofluorescence levels. C2Am was used at a concentration of 200 nM and AnxV at a concentration of 4 nM.

FLICA (fluorescent-labeled inhibitor of caspases) kit, which is based on caspase inhibitors that are linked to a green (carboxyfluorescein) fluorophore. FLICA is cell-permeant and is thought to bind only to active caspases within cells. Plots of SYTOX Green labeling as a function of NAD(H) autofluorescence were used, as before, to identify three cell populations: apoptotic cells (represented by the green symbols), late apoptotic/necrotic cells (orange symbols), and viable cells (blue symbols) (Figure 4). Dual scatter plots of drug-treated EL4 and MDA cells stained with FLICA and C2Am are shown in Figure 4b and g, respectively, and with FLICA and AnxV in Figure 4c and h, respectively. In EL4 cell populations treated with etoposide for 24 h, the MFIs for apoptotic and late apoptotic/necrotic cells stained with FLICA were up to 2-fold (P < 0.001) and 6-fold (P < 0.01) more, respectively, when compared with viable cells (Figure 4d). In MDA cells treated for 72 h with doxorubicin, these differences were up to 2-fold (P < 0.01) and 10-fold (P < 0.001) more for apoptotic and late apoptotic/necrotic cells, respectively, when compared with viable cells (Figure 4i). The binding of C2Am and AnxV to the viable cell population was examined by selecting the 5% of the viable cell population that showed the lowest FLICA labeling (Figure 4b,c and g,h) and analyzing the binding of C2Am and AnxV to this population. This is shown for EL4 cells in Figure 4e and for MDA cells in Figure 4j. This analysis confirmed that both C2Am and AnxV show low levels of binding to viable cells, which show the lowest FLICA labeling, but that AnxV binding to these cells was significantly greater than that of C2Am (P < 0.05, EL4 and P < 0.001, MDA). Effects of C2Am and AnxV Concentration on Their Binding to Drug-Treated Cells. The proteins were used at concentrations of between 0.05 and 1.0 µM for C2Am and 1-20 nM, for AnxV. The AnxV concentration recommended by the manufacturer is approximately 20 nM. Again, the three different cell populations, viable, apoptotic, and late apoptotic/necrotic, were selected based on SYTOX Green staining and NAD(H)

Figure 5. Effect of probe concentration on the specificity of labeling with C2Am and AnxV. C2Am was used at concentrations between 0.05 and 1.0 µM. AnxV was used at 50× lower concentrations, between 1 and 20 nM. EL4 cells were treated with etoposide (5 µM) for 16 h and MDA cells were treated with doxorubicin (1 µg/mL) for 72 h. MFI ratios for apoptotic/viable and necrotic/viable are shown for EL4 cells (a and b) and for MDA cells (c and d) following labeling with C2Am (open bars) or AnxV (closed bars). The different cell populations were identified based on their NADH autofluorescence and staining with SYTOX green, as in Figures 2-4. Data are shown as means ( SEM (n ) 3). The differences in MFI ratios between C2Am and AnxV were significant at *P < 0.05, plot b-0.1 and 0.2 µM; **P < 0.01, a-0.5 µM; ***P < 0.001, a-0.05 to 0.2 µM, c-0.05 to 1.0 µM, d-0.05 to 1.0 µM.

Detection of Cell Death Using C2A vs Annexin-V

autofluorescence. The levels of AnxV staining were consistently higher (P < 0.05) than C2Am for all cell populations (see Figure S4a-c in Supporting Information), above a concentration of 0.5 µM for C2Am and 10 nM for AnxV. This included the viable cell population, where the mean fluorescence intensity (MFI) for cells stained with AnxV was significantly higher (P < 0.01) than for those stained with C2Am (see Figure S4a in Supporting Information), despite AnxV being used at 50-fold lower concentrations. As a consequence, the mean fluorescence intensity (MFI) ratios for apoptotic/viable (Figure 5a) and necrotic/viable (Figure 5b) EL4 cells were significantly higher for C2Am than AnxV, at all the concentrations tested, indicating more specific labeling of both apoptotic (P < 0.01) and necrotic cells (P < 0.05) by C2Am. Similar results were obtained for the MDA line, for which the MFI of viable cells stained by AnxV was also significantly higher (P < 0.01) than for those stained with C2Am (see Figure S4d in Supporting Information), again despite being used at 50-fold lower concentrations. The levels of AnxV and C2Am staining of apoptotic and necrotic MDA cells were similar at these concentrations (see Figure S4e,f in Supporting Information), and like EL4 cells, the ratios of apoptotic/viable (Figure 5c) and necrotic/viable (Figure 5d) MDA cell staining were significantly higher for C2Am (P < 0.001), indicating again more specific labeling of both apoptotic and necrotic cells compared to viable cells. On the basis of the concentration dependency of C2Am binding to apoptotic EL4 (see Figure S4b in Supporting Information) and MDA cells (see Figure S4e in Supporting Information), which shows partial or total saturation of the MFI value, we estimated the concentration of PS exposed on these dying cells. Assuming a 1:1 binding model of C2Am to PS, this gave values of 277 ( 42 and 127 ( 10 pmol of PS per million apoptotic EL4 and MDA cells, respectively. Ca2+-Dependence of C2Am and AnxV Binding to Dying Cells. Binding of C2Am and AnxV to the PS exposed on dying cells is calcium-dependent. However, both proteins are also known to bind other plasma membrane phospholipids in a Ca2+-independent manner (21, 22). Drug-treated EL4 and MDA cells were incubated in the presence of Ca2+ (2 mM) or EDTA (10 mM) and binding of C2Am (200 nM) and AnxV (4 nM) was assessed by flow cytometry. The MFI ratios for binding to apoptotic versus viable and necrotic versus viable cells are shown in Figure 6. Binding to apoptotic EL4 cells was largely Ca2+-dependent for both proteins, and significantly higher than in the absence of Ca2+ (Figure 6a) (P < 0.001 and P < 0.05, for C2Am and AnxV, respectively). The mean MFI ratio for apoptotic/viable in the absence of Ca2+ was 0.04 and 0.12 for C2Am and AnxV, respectively. C2Am, however, showed a more pronounced Ca2+-independent binding to necrotic EL4 cells (Figure 6b) (a mean MFI ratio for necrotic/viable of 0.52 versus 0.16 for AnxV). This was also the case for necrotic MDA cells, where C2Am showed more Ca2+-independent binding (with a MFI ratio for necrotic/viable of 0.58 versus 0.08 for AnxV). However, in these cells, C2Am also showed relatively high levels of Ca2+-independent binding to apoptotic cells as well.

DISCUSSION Detection of PS with fluorescently labeled AnxV is a commonly used method for detecting apoptotic cell death in vitro (23, 24). Fluorescent, paramagnetic, and radionuclidelabeled derivatives of AnxV have also been used to detect cell death in vivo (25-30). In this study, we have evaluated, in comparison with AnxV, a novel probe for cell death detection based on the C2A domain of Synaptotagmin-I (11, 31). A GSTC2A fusion protein has been labeled previously with paramagnetic and radionuclide labels and used to detect apoptotic cells

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Figure 6. Effect of Ca2+ concentration on binding of C2Am and AnxV to apoptotic and necrotic cells. EL4 cells were treated with etoposide (5 µM) for 16 h and MDA cells were treated for 72 h with doxorubicin (1 µg/ml). MFI ratios for apoptotic/viable and necrotic/viable cells are shown for EL4 cells (a and b) and for MDA cells (c and d) following labeling with C2Am or AnxV in the presence of 2 mM Ca2+ (open bars) or 10 mM EDTA (filled bars). The different cell populations were identified based on their NADH autofluorescence and staining with SYTOX green, as in Figures 2-4. The ratios obtained in the absence of Ca2+ are also shown as a percentage of the ratios obtained in the presence of Ca2+. The data are shown as means ( SEM (n ) 3). *P < 0.05, ***P < 0.001.

in vivo using magnetic resonance and radionuclide imaging, respectively (13-15, 32). A problem with these early derivatives of C2A was that they were labeled on one or more of the ε-amino groups of the 35 lysine residues in the fusion protein, and thus, the imaging agent was a heterogeneous mixture of labeled molecules (15, 32). Moreover, in the case of the agent labeled with Gd3+-chelates, increased labeling led to a loss of affinity for PS and increased nonspecific binding (15). In this study, we have produced a site-directed mutant of C2A, containing a single cysteine residue C2A(S78C), which was labeled stoichiometrically with a fluorophore to yield a single chemical species (C2Am). The unmodified protein showed a comparable dissociation constant to the wild-type protein for PS (55 nM vs 20 nM, respectively) (16), and this was increased only slightly by modification of the cysteine residue with the fluorophore (Kd ) 71 nM). We have shown that this fluorescently labeled derivative was able to detect PS on apoptotic and necrotic murine lymphoma and human breast cancer cells in vitro by flow cytometry. Although the C2Am derivative showed less labeling of apoptotic and necrotic cells than a similarly labeled commercial preparation of AnxV, the C2Am derivative showed lower binding to viable cells, despite being used at 50-fold higher concentrations and, as a consequence, better specificity for the detection of apoptotic and necrotic cells. This was reflected in the consistently higher ratios for the apoptotic/viable and necrotic/viable mean fluorescence intensities (MFI) for cells labeled with C2Am as compared with AnxV. Titration of apoptotic EL4 and MDA cells with increasing concentrations of C2Am led to saturation of the bound fluorescence intensity and enabled us to estimate the abundance of PS, which for EL4 was 277 ( 42 pmol/106 cells and for MDA 127 ( 10 pmol/106 cells. The concentration of PS on EL4 cells is comparable to the level reported for apoptotic Jurkat cells of 240 pmol/106 cells, which is a human lymphoid cell line (33). In order to investigate further the apparent binding of AnxV to viable cells, we used a fluorescently labeled inhibitor of

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activated caspases (FLICA) to confirm that this cell population, which was identified as viable from measurements of NADH autofluorescence, was indeed viable. There has been some discussion as to whether FLICA detects activated caspases or the cell damage mediated by activated caspases (34, 35). Regardless of the mechanism responsible, we expect viable cells to show only low levels of FLICA staining. The viable cell population, identified from measurements of NADH autofluorescence in drug-treated cell preparations, showed 2-fold and 6-fold less labeling with FLICA, when compared with apoptotic and late apoptotic/necrotic cells, respectively, in EL4 cells and 2-fold and 10-fold less labeling, respectively, in MDA cells, confirming that this was a genuinely viable cell population. When we analyzed C2m and AnxV binding to the 5% of the viable cell population that showed the lowest labeling with FLICA, there was considerably more labeling with AnxV (up to 5-fold) than with C2Am. The Ca2+-dependent binding of C2A and AnxV to PS is well-documented (12, 23). Binding of both probes to the apoptotic cell fraction was largely quenched (60-99%) by the addition of EDTA (10 mM). This was also the case for AnxV binding to necrotic cells (8-16% of Ca2+-independent binding). However, a large component of C2Am binding to necrotic cells was Ca2+-independent (53% for EL4 and 58% for MDA). Since there is no evidence that C2A has any detectable affinity for PS in the absence of Ca2+ (36), the reason for this binding is unclear, although it could relate to the smaller size of C2Am making it better able to cross the ruptured cell membranes of necrotic cells. The Ca2+-independent binding observed for both proteins may be due to binding to other phospholipids that are externalized during apoptosis (e.g., phosphatidylethanolamine or phosphatidylinositol) (37). Although both proteins exhibit Ca2+-dependent binding of anionic phospholipids, their three-dimensional structures, mechanisms of binding, and ultimately their affinities for PS differ appreciably. The C2A domain is a compact eight-stranded β-sheet structure interconnected by seven loops (31). The Ca2+ and PS binding sites are formed by the three loops at the top of the domain. There are three sites for Ca2+ in the Ca2+ binding pocket of C2A, and this binding stabilizes the loops but produces no significant conformational changes (38). The empty coordination sites on the bound Ca2+ ions are filled by direct interaction with anionic phospholipids. In contrast, AnxV is composed of four compact R-helical domains. Each domain contains a Ca2+ and phospholipid binding site (39). Two Ca2+ ions are bound at each binding site (40) and Ca2+-induced conformational changes allow these multiple PS binding sites to interact in a cooperative manner, giving rise to the high affinity for PS (41). Furthermore, chemical cross-linking studies have shown that Annexin-V can form calcium-induced trimers, hexamers, and larger aggregates on phospholipid membranes (42). This multivalent binding likely explains the very high affinities reported for AnxV binding to PS, in the range 0.03-15 nM (43, 44). This range is considerably lower than that reported previously for C2A (16) and that measured here. The much higher affinity of AnxV for PS may explain the binding of this protein to viable cells, which could express low levels of PS. The lower affinity of C2Am for PS may reduce binding to viable cells and hence explain why it is a more specific probe for cell death detection than AnxV.

CONCLUSIONS We have synthesized a chemically well-defined fluorescent derivative of a site-directed mutant of the C2A domain of Synaptotagmin-I (S78C), which shows lower binding to viable cells than a similarly labeled Annexin V derivative and consequently has better specificity for detecting cell death. The

Alam et al.

lower binding to viable cells may be due to the lower affinity of C2A for PS. The lower binding of C2A to viable cells may be important when radionuclide derivatives of this protein are used to detect cell death in vivo, since this may reduce the nonspecific tissue accumulation observed with radionuclidelabeled derivatives of Annexin-V.

ACKNOWLEDGMENT The work was supported by a Cancer Research UK Programme grant (to K.M.B.; C197/A3514) and by a Translational Research Program Award from The Leukemia & Lymphoma Society. T.H.W. was in receipt of BBSRC CASE studentship with GE Healthcare. We thank Dr. Len Packman at the Department of Biochemistry, University of Cambridge, for mass spectrometric analysis and Dr. Maaike de Backer, also at the Department of Biochemistry, for suggesting candidate mutations. Supporting Information Available: Expression and purification of C2A(S78C), the protein assay, electrospray ionization (ESI) mass spectrometry, and surface plasmon resonance (SPR) measurements are described in more detail. There is a table showing the dissociation constants derived from affinity and kinetic analysis of the SPR data, a figure showing the ESI-mass spectrum of the C2Am-AlxF647 conjugate, figures showing the SDS-PAGE gels of the C2Am-AlxF647 and AnxV-AlxF647 conjugates and a figure demonstrating the effect of probe concentration on the specificity of labeling with C2Am and AnxV. This material is available free of charge via the Internet at http://pubs.acs.org.

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