A novel bicistronic sensor vector for detecting caspase-3 activation.

JPM-06235; No of Pages 8 Journal of Pharmacological and Toxicological Methods xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Pharmacological and Toxicological Methods journal homepage: www.elsevier.com/locate/jpharmtox

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Brief communication

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A novel bicistronic sensor vector for detecting caspase-3 activation a

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Article history: Received 6 August 2014 Accepted 28 November 2014 Available online xxxx

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Keywords: Caspase-3 activation Caspase-3 sensor Cell-based assay

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Introduction: Apoptosis is involved in pathological cell death of a wide range of human diseases. One of the most important biochemical markers of apoptosis is activation of caspase-3. Ability to detect caspase-3 activation early in the pathological process is important for determining the timing for interfering with apoptosis initiation and prevention of cell damage. Techniques allowing detection of caspase-3 activity at a single cell level show increased sensitivity, compared to biochemical assays; therefore, we developed a novel bicistronic caspase-3 sensor vector enabling detection of caspase-3 activity in individual cells. Methods: We employed green fluorescent protein (GFP) as a reporter for caspase-3 activation in our constructs and assessed the functionality of the generated constructs in transiently transfected Neuro2A and HEK293 cells under basal conditions and following application of okadaic acid (OA) or staurosporine (STS) to induce apoptosis. To ensure responsiveness of the new sensor vector to active caspase-3, we co-transfected the sensor with plasmid(s) overexpressing active caspase-3 and quantified GFP fluorescence using a plate reader. Results: We observed an increase in GFP expression in cells transfected with the new bicistronic caspase-3 sensor in response to both OA and STS. We also showed a significant increase in GFP fluorescence intensity in cells co-expressing the sensor with the plasmid(s) encoding active caspase-3. Discussion: We generated a novel bicistronic caspase-3 sensor vector which relies on a transcription factor/ response element system. The obtained sensor combines high sensitivity of the single cell level detection with the possibility of automated quantification. © 2014 Published by Elsevier Inc.

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Centre for Brain Research, University of Auckland, 85 Park Rd, Grafton, Auckland 1142, New Zealand Dept. of Molecular Medicine & Pathology, University of Auckland, 85 Park Rd, Grafton, Auckland 1142, New Zealand Dept. of Pharmacology & Clinical Pharmacology, University of Auckland, 85 Park Rd, Grafton, Auckland 1142, New Zealand

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Tatyana Vagner a,b,⁎, Alexandre Mouravlev b,c, Deborah Young b,c

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1. Introduction

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Caspase-dependent apoptosis plays a key role in the pathogenesis of a large number of human diseases, including multiple types of cancer, neurological disorders, cardiovascular disorders, and autoimmune diseases (reviewed in (Favaloro, Allocati, Graziano, Di Ilio, & De Laurenzi, 2012)). Activation of caspase-3 has been identified as a central event in the process of apoptosis (Budihardjo, Oliver, Lutter, Luo, & Wang, 1999; Thornberry & Lazebnik, 1998; Wolf & Green, 1999). Thus, measurement of caspase-3 activity is essential in studies aimed at developing therapeutic approaches based on regulation of apoptosis. Standard caspase-3 assays based on peptide substrates labelled with cleavable fluorophores have been widely used for determining caspase3 activity from cellular lysates (Gurtu, Kain, & Zhang, 1997; Wang, Liao, & Diwu, 2005). However, their short wavelength, low extinction coefficient and high fluorescent background result in low assay sensitivity. Also, a few studies revealed that cell population based biochemical

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⁎ Corresponding author at: Centre for Brain Research, University of Auckland, 85 Park Rd, Grafton, Auckland 1142, New Zealand. Tel.: +64 09 3737599. E-mail address: [email protected] (T. Vagner).

caspase-3 assays suggest that protease activation is more delayed than when observed on a single cell level (Rehm et al., 2002; Tyas, Brophy, Pope, Rivett, & Tavare, 2000; Werner & Steinfelder, 2008). Compared to biochemical assays, single cell based assays are less invasive, provide an increased sensitivity and allow monitoring of caspase-3 activity in live cells. Werner and Steinfelder (2008) established a fluorescence microscopy technique for detection of caspase-3 activation at the single cell level. This technique relied on a caspase-3 sensor that expressed a fusion protein containing a fluorescent EYFP (enhanced yellow fluorescent protein) coupled to a nuclear localization signal. Cleavage of the fusion protein at a sequence recognized by caspase-3 allowed EYFP to translocate into the nucleus of apoptotic cells and these fluorescently labelled nuclei could be visually detected and quantified by an investigator. This approach demonstrated significantly increased accuracy compared to the biochemical assay, especially at earlier time points and at lower concentrations of pro-apoptotic agents. We set out to develop a sensitive technique that would allow detection of caspase-3 activation in cells at the very early stages of apoptosis that could be applicable for both in vitro and in vivo applications. In this study, we first generate and test a reporter construct for caspase-3 activation in the context of a recombinant adeno-associated

http://dx.doi.org/10.1016/j.vascn.2014.11.006 1056-8719/© 2014 Published by Elsevier Inc.

Please cite this article as: Vagner, T., et al., A novel bicistronic sensor vector for detecting caspase-3 activation, Journal of Pharmacological and Toxicological Methods (2014), http://dx.doi.org/10.1016/j.vascn.2014.11.006

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A plasmid encoding a rAAV expression cassette, consisting of a hybrid cytomegalovirus enhancer/chicken β-actin (CAG) promoter, polylinker sequence (pL), woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and bovine growth hormone polyadenylation signal (BGHpA), flanked by AAV2 inverted terminal repeats was used for generating the constructs employed in this study. An enhanced Aequorea victoria green fluorescent protein (eGFP) was PCR-amplified from the pIRES-EGFP plasmid (Clonetech) using the following primers: 5′-ATACTCGAGCGCCACCATGGTGAGCAAG-3′ (forward) and 5′-ATAACGCGTCTTGTACAGCTCGTCCATG-3′ (reverse). Custom synthetic oligonucleotides encoding a nuclear localisation signal of the SV40 large T-antigen (Kalderon, Roberts, Richardson, & Smith, 1984) were obtained from Life Technologies™. A 36-nucleotide sequence encoding the region of Poly (ADP-ribose) polymerase (PARP) cleaved by caspase-3 coupled to the nuclear export signal of the MAPKK (Henderson & Eleftheriou, 2000) was custom synthesized by GenScript (USA). A chimeric transcription factor consisting of the DNA-binding domain of the auxin response factor 5 (ARF5) fused to the VP16 activation domain and the ARF5 response element (AuxRE) (Tiwari, Hagen, & Guilfoyle, 2003) were generously provided by Tom Guilfoyle, Department of Biochemistry, University of Missouri, Columbia, MO, USA. The sequences encoding the large and the small subunits of active caspase-3 (GenBank: NM_012922) were custom synthesized by Blue Heron® (USA). Using standard molecular cloning techniques, the PCR products and the synthetic DNA fragments were cloned into the pL of the rAAV expression cassette plasmid to generate the constructs used in this study.

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2.2. Cell culture

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Human embryonic kidney 293 (HEK293) cells and mouse neuroblastoma Neuro2A cells were maintained as monolayer in 75 cm2 flasks (Nunc) at 37 °C in 5% CO2. HEK293 were maintained in pre-made Dulbecco's modified Eagle's medium (DMEM, Life Technologies™) supplemented with 10% foetal bovine serum (FBS, HyClone™), 1 mM sodium pyruvate (Life Technologies™), and 100 μM non-essential amino acids (Life Technologies™). Neuro2A were maintained in complete Eagle's minimal essential medium prepared using MEM, NEAA powder (Life Technologies™) and supplemented with 10% FBS (HyClone™), 1 mM sodium pyruvate (Life Technologies™), and 26 mM NaHCO3. Pre-made Iscove's Modified Dulbecco's Medium (IMDM, Life Technologies™) supplemented with 5% FBS (HyClone™) was used for transfecting HEK293 cells.

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2.3. Transfection

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HEK293 cells were transfected using a calcium phosphate transfection protocol. Cells were plated at a density of 1 × 105/well onto 24 well plates, 24 h prior to transfection. The following day the medium was aspirated and replaced with pre-warmed complete IMDM 2–3 h prior to transfection. In a sterile tube, 0.25 μg plasmid DNA per well was mixed with an appropriate volume of 0.25 M CaCl2 in sterile H2O. An equal amount of 2xHeBS (HEPES-buffered saline) was added to the DNA–CaCl2 mixture while pipetting up and down vigorously. After

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Primary polyclonal rabbit antibody that recognizes cleaved caspase-3 (Asp175) (Cell Signalling Technology) was applied at the 1:1000 dilution to each well in 200 μl of immunobuffer (4% horse serum (Invitrogen), 0.04% thiomersal (Sigma) in 1x PBS-Triton) and incubated overnight at room temperature. Following the incubation, cells were washed twice in PBS-triton and anti-rabbit IgG-Cy3 (1:250, Jackson laboratory) applied. After 5–6 h of incubation at room temperature, the cells were washed extensively with PBS-triton and analysed. Immunolabelling as well as native GFP fluorescence was examined using an inverted ECLIPSE TE-2000S microscope with an epifluorescent attachment (Nikon).

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incubation at room temperature for 5 min, 80 μl of mixture was pipetted dropwise into each well. After 5–6 h, the medium was aspired and replaced with pre-warmed complete DMEM. Forty-eight hours after transfection the cells were fixed by incubation in 400 μl of 10% neutral buffered formaldehyde (Sigma) for 15–20 min, rinsed in 1xPBS containing 0.2% (v/v) Triton X100 (PBS-triton) 2 × 5 mins and native GFP fluorescence was examined using an inverted ECLIPSE TE-2000S microscope with an epifluorescent attachment (Nikon). Neuro2A cells were transfected in 24 well plates using FuGENE® HD reagent (Roche) according to the manufacturer's protocol. Twenty-four hours after transfection, the media was aspirated and the cells were fixed in 10% neutral buffered formaldehyde, rinsed twice in PBS-triton and analysed by fluorescence microscopy, as above.

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2.5. Quantification of GFP fluorescence intensity and statistical analysis

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The GFP fluorescence intensity was measured 48 h after transfection in the EnSpire® Multimode plate reader (Perkin Elmer Inc.) at Ex = 488 nm and Em = 509 nm, using bottom read-based scan throughout the entire well area (n = 3). The data from three independent experiments were analysed using one-way ANOVA with post-hoc Tukey test, with significance level set at P b 0.05.

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3. Results

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In order to generate a reporter construct that could be used for detection of caspase-3 activity and could facilitate identification of the cells at the early stages of apoptosis, we first employed the vector design similar to that of the Clontech vector used by Werner and Steinfelder. However, since we planned to use our caspase-3 reporter construct both in vitro and in vivo, unlike the caspase-3 sensor employed by Werner and Steinfelder, our construct was incorporated in a plasmid containing a rAAV expression cassette. The construct encoded a green fluorescent protein (GFP) fused with a nuclear localization signal (NLS, (Kalderon et al., 1984)) and linked to a dominant nuclear export signal (NES, (Henderson & Eleftheriou, 2000)) via a caspase-3 cleavage site (pGFP-NLS-Casp-NES). Because the NES dominates the NLS, the fulllength fluorescent fusion protein distributes to the cytosol under basal conditions. However, when caspase-3 is rendered active, the NES should be cleaved off from the fusion protein allowing the GFP-NLS to translocate to the nucleus via NLS. Thus, this construct can be used as a reporter vector to analyse caspase-3 activity at a cellular level by visualization of the GFP redistribution from the cytosol to the nucleus of the cells. Also, a control construct for nuclear localization of GFP, pGFP-NLS, was generated by eliminating the sequence encoding the caspase-3 cleavage site and the NES from the original reporter construct (Fig. 1.) The functionality of the caspase-3 reporter construct was analysed by visualization of native GFP fluorescence in transiently transfected Neuro2A or HEK293 cells upon induction of apoptosis with okadaic acid (OA) or staurosporine (STS). A plasmid expressing an unaltered, original, GFP (pGFP) under control of the CAG promoter was used as an additional control to monitor the transfection efficiency as well as

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virus (rAAV) expression cassette employing a vector design similar to one used in the study by Werner and Steinfelder. We next develop and evaluate the functionality of a novel bicistronic caspase-3 sensor vector that allows machine-based quantification of the GFP readout but maintains the sensitivity of a single cell detection method.

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Fig. 1. Reporter construct for caspase-3 activation. Under normal conditions, the NES should restrict distribution of the GFP-NLS fusion protein in the cell to the cytosol. Upon activation of caspase-3, the NES should be cleaved off and the GFP-NLS should translocate to the nucleus. Thus, GFP-positive nuclei would indicate activation of caspase-3 in the cells. CAG, hybrid cytomegalovirus enhancer/chicken β-actin promoter; GFP, green fluorescent protein; NLS, nuclear localization signal; NES, nuclear export signal.

Fig. 2. Translocation of the GFP-NLS chimeric protein to the nucleus in Neuro2A cells treated with OA or STS. (A) Under basal conditions, GFP fluorescence was observed predominantly in the cytosol of the transfected cells. To induce caspase-3 activation, cells were treated with either 30nM OA (B) or 0.7 μM STS (C). Twenty-four hours after the treatment, GFP signal could be detected in both nucleus and cytoplasm of the cells. However, in both OA- (D) and STS-treated (E) samples, in a number of cells GFP localized only in the cytosol (showed with the arrows). (F) Cells transfected with the nuclear localization control vector, pGFP-NLS, in which chimeric GFP-NLS protein lacked the NES. (G). Cells transfected with the pGFP encoding an unaltered GFP. Scale bar: 50 μm.


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transcription factor ARF5 fused to the herpes virus minimal transcriptional activation domain VP16 (referred to as ‘ARF5’ hereafter). The chimeric transcription factor was coupled to the dominant NES via a linker containing a cleavage site for caspase-3. Cistron 2 consisted of the GFP reporter gene positioned downstream from the ARF5 response element (AuxRE). It was hypothesized that under basal conditions, the dominant NES should restrict the ARF5 to the cytosol and reporter gene expression should be quiescent. However, when caspase-3 is activated, the NES is cleaved from the ARF5, allowing the ARF5 to translocate into the nucleus. In the nucleus, the ARF5 binds to the AuxRE within the cistron 2 and drives the transcription of the GFP transgene (Fig. 3). Thus, GFP expression is detectable only in the presence of caspase-3 activity and can be quantified by measuring fluorescence intensity using a plate reader. To see if the ARF5/AuxRE system was functional in mammalian cells and the GFP reporter could be activated in response to caspase-3 activation, the ARF5/AuxRE sensor vector was first tested in transiently transfected HEK293 cells. Twenty-four hours post-transfection, cells were challenged with either OA or STS to induce apoptosis and GFP expression was visualized by fluorescent microscopy 24 h later. To control for leaky and/or non-specifically activated GFP expression from the AuxRE, a control construct lacking the ARF5, ΔAuxRE, was also included. GFP expression was negligible in the cells transfected with the ΔAuxRE construct lacking the ARF5 (Fig. 4, top row), suggesting that non-specific inducibility of the AuxRE was minimal. Low-level GFP expression was observed in some cells transfected with the caspase-3 sensor vector under the basal conditions (Fig. 4, bottom row, vehicle), indicating that there might have been low caspase-3 activity in those cells. Following the application of either OA or STS, a clear increase in GFP fluorescence was observed (Fig. 4, bottom row, OA and STS). This result demonstrated that the reporter gene expression in transfected cells was activated in response to exposure to the apoptosis-inducing drugs. Although both OA and STS have been shown to induce caspase-3 activation (Allan et al., 2003; Chopp, Li, & Jiang, 1999; Koh et al., 1995; Nuydens et al., 1998; Suuronen, Kolehmainen, & Salminen, 2000), a cellular response to these drugs can be cell line-specific: both OA and STS have been reported to induce apoptosis via a caspase-3independent pathway in some cell lines (Kitazumi et al., 2010; Zhang, Gillespie, & Hersey, 2004). To ensure that the created sensor vector

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‘natural’ pattern of intracellular distribution of the overexpressed GFP, when it is not affected by fusion with either NLS or NES. Twenty-four hours post-transfection, Neuro2A and HEK293 cells were exposed to OA or STS to induce apoptosis. The distribution of GFP within the cells was analysed 24 h post-treatment by fluorescence microscopy. In cells transfected with the pGFP-NLS-Casp-NES caspase-3 reporter, GFP fluorescence was detected predominantly in the cytosol under basal conditions in both cell lines (Fig. 2, A). After exposure to OA or STS, GFP fluorescence in many cells was detected in both the nucleus and cytosol, suggesting that in some cells, the NES had been removed from the fusion protein due to activation of caspase-3 allowing GFP-NLS to translocate to the nucleus (Fig. 2, B, C). However, in the toxin-treated samples, there were also many cells that exhibited a phenotype similar to that of non-toxin-treated cells—in which GFP was localized only in the cytosol (Fig. 2, D, E). This was also observed by Werner and Steinfelder in their study which employed a commercial caspase-3 reporter construct (Werner & Steinfelder, 2008). It is of interest that in cells transfected with the nuclear localization control plasmid, pGFP-NLS, GFP fluorescence could be detected both in the nucleus and in the cytosol (Fig. 2, F). Similar GFP distribution pattern was observed in the cells expressing the unaltered GFP, without the NLS, from the pGFP, suggesting that this cell distribution pattern is characteristic of the original protein (Fig. 2, G). Therefore, given the tendency of the GFP to naturally localize in the cytoplasm, as well as its stability and excessive amounts in the cell produced by overexpression, the NLS used might not have been strong enough to restrict GFP-NLS to the nucleus. Due to the complex nature of the NES/NLS interaction within the fusion protein molecule, the signal-to-noise (signal:noise) ratio was very low in this system, making the required time-consuming procedure of visual cell counting even more laborious. Therefore, although the generated caspase-3 reporter construct was demonstrated to be indicative of caspase-3 activation at the level of individual cells, this detection system would certainly benefit from further optimization. To further optimize this system to increase the signal:noise ratio to enable easier detection and quantification of caspase-3 activation, we created a novel bicistronic caspase-3 sensor vector which relies on a transcription factor/response element system. Within this system, cistron 1 of the sensor vector encoded a chimeric transcription factor consisting of the DNA-binding domain of the plant

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Fig. 3. A novel bicistronic caspase-3 sensor vector. Under basal conditions, the dominant NES restricts the ARF5 to the cytosol and the ARF5 is therefore unable to bind to the AuxRE controlling the expression of the reporter gene. However, when caspase-3 is activated, the NES is cleaved off and the ARF5 translocates to the nucleus, binds to the AuxRE and drives the transcription of the GFP reporter. Thus, the GFP is produced only when caspase-3 is active. ARF5, chimeric transcription factor consisting of the DNA-binding domain of the auxin response element 5 fused to the herpes virus minimal transcriptional activation domain VP16; NES, nuclear export signal; CAG, hybrid cytomegalovirus enhancer/chicken β-actin promoter; AuxRE, ARF5 response element; GFP, green fluorescent protein.


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Fig. 4. Activation of the GFP reporter within the ARF5/AuxRE caspase-3 sensor vector in HEK293 cells treated with OA or STS. Top row: Representative images showing the negligible basal GFP expression in the cells transfected with control plasmid ΔAuxRE lacking the ARF5. Bottom row: Representative images showing the increase in GFP expression in the cells transfected with the ARF5/AuxRE caspase-3 sensor vector, 24 h after treatment with 30nM OA or 0.7 μM STS. Scale bar: 250 μm.

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regions that would eliminate the protein-coding sequences, two different promoters were used to drive the transcription of the active caspase-3 subunits. The large subunit was expressed from the weaker promoter NSE(300) (3′-terminal 310 bp of neuronal-specific enolase promoter), and the small subunit was expressed from the strong CAG promoter (Fig. 5, B). We co-expressed the ARF5/AuxRE sensor vector, or the ΔAuxRE construct lacking the ARF5, with either pCaspL − CaspS, or pCaspL + pCaspS plasmids by transient transfection of HEK293 cells. To equalize the total amount of plasmid DNA used for transfection in control cells, a plasmid overexpressing an inert protein (Cre) was used (referred to as ‘control’). The presence of the active caspase-3 in the transfected cells was confirmed immunocytochemically, using the antibody against active caspase-3 (Fig. 6, A). GFP fluorescence was analysed 48 h post-

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was specifically responsive to caspase-3 activation, we developed a cell model in which caspase-3 was constitutively active. The active caspase-3 molecule has a modular structure that is self-assembled in the cell and comprised of two large and two small subunits (Fig. 5, A). Therefore, to create a cell model for testing the ARF5/AuxRE sensor vector, plasmids expressing the subunits of active caspase-3 were used. In our model, the large and the small subunits of active caspase-3 were expressed either from two plasmids, each encoding one of the subunits, or from a single plasmid, encoding both subunits. In the two-plasmid system (pCaspL + pCaspS), each subunit was expressed from the strong hybrid cytomegalovirus enhancer/chicken β-actin (CAG) promoter. In the case of the single plasmid system (pCaspL − CaspS), the construct contained two cistrons positioned in the “tail-to-tail” orientation. To avoid a possible recombination between the promoter

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Fig. 5. Formation of active caspase-3 enzyme from inactive proenzyme and the plasmids used to express the subunits of active caspase-3. (A) Caspase-3 molecules are found in cells as preformed inactive dimers, in which the large and the small subunits covalently connected via the intersubunit linkers. The inactive state is maintained by steric hindrances, which are imposed by these intersubunit linkers and result in misalignment of active sites within the dimer. Proteolitic cleavage of the linkers permits the formation of functional active sites within the enzyme. (B) Schemes of the two-plasmid (pCaspL + pCaspS) and one-plasmid (pCaspL − CaspS) systems used to generate a cell model of constitutively active caspase-3. CAG, hybrid cytomegalovirus enhancer/chicken β-actin promoter; Caspase-3 L, sequence encoding the large subunit of active caspase-3; Caspase-3 S, sequence encoding the small subunit of active caspase-3; NSE(300), 3′-terminal 310 bp of neuronal-specific enolase promoter.


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Fig. 6. Activation of the GFP reporter within the ARF5/AuxRE caspase-3 sensor vector in response to active caspase-3. (A) Anti-caspase-3 immunofluorescent staining of HEK293 cells co-transfected with the ARF5/AuxRE caspase-3 sensor vector and either control construct, or one (pCaspL − CaspS), or two (pCaspL + pCaspS) plasmids expressing active caspase-3. Scale bar: 50 μm. (B). In the cells co-expressing the AFR5/AuxRE sensor vector (black bars on the graph) and one plasmid pCaspL − CaspS encoding both subunits of active caspase-3, GFP fluorescence was significantly increased compared to the cells co-expressing the AFR5/AuxRE sensor vector and the control plasmid. In contrast, in the cells co-expressing the AFR5/AuxRE sensor vector and two plasmids pCaspL + pCaspS encoding one subunit of active caspase-3 each, GFP fluorescence was decreased. The level of basal GFP fluorescence from the ΔAuxRE construct lacking the ARF5 (grey bars on the graph) was not affected in either one- (pCaspL − CaspS) or two-plasmid (pCaspL + pCaspS) systems. Scale bar: 250 μm. On the graph, values represent the mean +/− SEM of three independent experiments, *p b 0.05, ****p b 0.0001.

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transfection by fluorescent microscopy and quantified using a plate reader. A significant increase in GFP fluorescence was detected when the AFR5/AuxRE sensor vector was co-expressed with the single plasmid encoding both subunits of the active caspase-3, pCaspL − CaspS, compared to cells co-transfected with the AFR5/AuxRE sensor vector and the control plasmid (Fig. 6, B). This confirmed that the observed increase in GFP fluorescence intensity was specific to caspase-3 activation in the cells. When the two-plasmid system, pCaspL + CaspS, was used to coexpress the active caspase-3 with the AFR5/AuxRE sensor vector, a

significantly reduced level of GFP fluorescence was observed (Fig. 5, B), concomitant with the development of apoptotic morphology in a large portion of the cells, with some of the cells detaching from the surface of the culture dish (data not shown). This meant that the level of the active caspase-3 produced by the two-plasmid system was toxic.

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Activation of the death protease caspase-3 is a central event in the 319 process of apoptosis (Budihardjo et al., 1999; Thornberry & Lazebnik, 320 1998; Wolf & Green, 1999) and leads to the characteristic morphological 321



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model of apoptosis. In one variant of this model, the large and the small subunits of active caspase-3 were expressed from the individual plasmids each under control of the strong CAG promoter. In the other variant, both subunits were expressed from one plasmid: the small subunit under control of the CAG promoter, the large subunit under control of the weaker NSE(300) promoter. Using the one-plasmid apoptosis model, we demonstrated a significant increase in GFP fluorescence and confirmed that it was specific to caspase-3 activation. In the two-plasmid apoptosis model, however, a decrease in GFP fluorescence accompanied by severe apoptotic morphology of a large portion of the cells was observed. Severe apoptotic morphology of the cells in which expression of both caspase-3 subunits was driven by the strong CAG promoter suggested that the level of protease activity achieved by the two-plasmid system was too high and toxic. Thus, a markedly reduced GFP fluorescence intensity can be explained by the general ‘shut down’ of the cellular synthetic processes associated with apoptosis (reviewed in (Clemens, Bushell, Jeffrey, Pain, & Morley, 2000)). Another possible explanation for the observed decrease in the GFP fluorescence is that excessive amount of the protease indiscriminately cleaved proteins within the cells, including the overexpressed GFP. Meanwhile, the level of caspase-3 activity produced by the oneplasmid system, in which the expression of the large subunit was driven by the weaker NSE(300) promoter, appeared to be elevated enough to induce the increase in the reporter gene expression, but still physiologically tolerable, at least at the time point examined. In conclusion, a novel sensitive molecular tool for assaying caspase-3 activity in live cells was developed. The new AFR5/AuxRE caspase-3 sensor vector allows detection of the increased caspase-3 activity in individual cells early on in the apoptotic process, before the onset of morphological alterations. The approach involving the use of our AFR5/AuxRE caspase-3 sensor vector followed by the measurement of the GFP fluorescence intensity in the whole cells combines the high sensitivity of the single cell level detection with the possibility of automatized quantification of the signal. There are a few similar approaches that also allow measurement of caspase-3 activity at the single cell level which are based on fluorescence resonance energy transfer (FRET). The FRET-based caspase-3 sensors express two differently coloured fluorescent proteins, a donor and an acceptor, which are fused via a linker containing a caspase-3 cleavage site. The emission spectrum of the donor overlaps with the excitation spectrum of the acceptor, thus, placing them in close proximity (i.e. b5 nm) to each other within the sensor allows FRET between the two fluorescent moieties to occur. Under basal conditions, when the donor is excited, fluorescence from the acceptor can be detected, but upon cleavage of the linker by caspase-3, the fluorescence from the donor is detected. Thus, the FRET-based techniques allow monitoring of the kinetics of caspase-3 activation in living cells during apoptosis. However, application of this method has a number of limitations, such as a relatively low dynamic range (donor/acceptor emission ratio change), which is, in turn, limited by FRET efficiency, and problematic spectral separation due to a significant cross-talk between the FRET partners (Piston & Kremers, 2007). In addition, quantification of FRET can be complicated by technical limitations of available microscopy software and hardware or can require usage of costly equipment or data analysis is time-consuming (e.g. when FRET is quantified by fluorescence lifetime imaging microscopy (FLIM) or multi-parameter flow cytometry) (Bacskai, Skoch, Hickey, Allen, & Hyman, 2003; Shcherbo et al., 2009; Wang, Chen, Qu, & Wei, 2010; Wu et al., 2006). The ARF5/AuxRE caspase-3 sensor vector developed in the current work offers a simpler technique that circumvents the above limitations of the FRET-based approaches. The use of the single fluorescent protein enables simple and reliable detection of the signal, which can be quantified using standard equipment. Thus, the approach reliant on the bicistronic ARF5/AuxRE system can be used both as an alternative and/ or complementary approach to the FRET-based techniques.

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cellular changes associated with it (Janicke, Sprengart, Wati, & Porter, 1998). Since the most commonly used protease assays measure caspase-3 activity in the lysate of a whole population of treated cells, sensitivity to identify drug-induced apoptosis might be reduced with this kind of assay. The use of the caspase-3 sensor vector followed by the fluorescence microscopy analysis enables detection of caspase-3 activity in individual cells with increased sensitivity, compared to biochemical assays (Rehm et al., 2002; Tyas et al., 2000; Werner & Steinfelder, 2008). We set out to develop an approach that would allow detection of caspase-3 activity before the onset of the irreversible physiological changes leading to cell death in vitro. To address this need, we first generated a caspase-3 reporter construct in the context of the rAAV expression cassette to be used in conjunction with the microscopic technique established by Werner and Steinfelder (2008). This construct expressed a fusion protein containing the GFP coupled to the NLS and linked to the dominant NES via the caspase-3 cleavage site. With this construct, activation of caspase-3 could be visualized by translocation of the fluorescent fusion protein from the cytosol to the nucleus. When analysed in transiently transfected cells, the generated reporter construct was shown to be indicative of caspase-3 activation in response to OA or STS—the cells displayed labelled nuclei upon treatment with either of these apoptosis inducers. However, the signal:noise ratio was very low in this system due to the complex nature of the NES/NLS interaction within the fusion protein molecule. In the cells expressing the nuclear localization control construct, as well as in the cells where the GFP-NLS had translocated to the nucleus upon exposure to OA or STS, the fluorescent fusion protein was observed both in the cytosol and in the nucleus. A few reasons can account for the mixed intracellular localization of the fluorescent fusion protein. The chosen NLS might not be strong enough to keep the GFP-NLS within the nucleus, given the natural tendency of the GFP to distribute in the cytoplasm of the cell, together with its stability and excess amount due to overexpression. Furthermore, the relatively small size of the GFP-NLS fusion protein (≈42 kDa) can allow it to randomly diffuse through the nuclear membrane, therefore, although it does translocate to the nucleus due to the presence of the NLS, its accumulation in the nucleus would require protein import to be constitutively active. Hence, GFP fluorescence could occur in both cellular compartments. Moreover, the nuclear protein import pathway can be inhibited by a variety of stress signals (Stochaj, Rassadi, & Chiu, 2000), which could also contribute to the leakage of the GFP-NLS to the cytosol after it had translocated to the nucleus upon induction of apoptosis. In order to generate a more reliable reporter, we developed a novel bicistronic caspase-3 sensor vector reliant on the ARF5/AuxRE transcription factor/response element system. The ARF5 is fused via the caspase-3 cleavage site to the dominant NES and thus restricted to the cytosol. After cleavage of the fusion protein by caspase-3 at its recognition site, the ARF5 translocates to the nucleus and drives the expression of the GFP reporter via the AuxRE. One of the advantages of this new caspase-3 sensor vector is that protease activity in the cells is much easier to detect: the fluorescent signal is almost completely absent under basal conditions and appears only when caspase-3 is activated, as opposed to being present at all times and shuttling between the cellular compartments. Another advantage of the ARF5/AuxRE caspase-3 sensor is that it allows quick automatized quantification of the GFP fluorescence using a plate reader, as opposed to time-consuming visual count of labelled nuclei by the investigator. We showed that the GFP expression in the cells transiently transfected with the ARF5/AuxRE caspase-3 sensor vector was activated in response to exposure to the apoptosis-inducing agents, OA or STS. However, as OA- or STS-induced apoptosis can sometimes occur via a caspase-3-independent pathway (Kitazumi et al., 2010; Zhang et al., 2004), to ascertain that the observed increase in the GFP fluorescence resulted from caspase-3 activation, we developed a novel cellular

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This work was supported by a University of Auckland International Doctoral Scholarship (T. Vagner) and funding from the Royal Society Marsden Fund, UOA0714, and New Zealand Health Research Council, 10/149 (D. Young).

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Further studies using the ARF5/AuxRE caspase-3 sensor vector can provide valuable insight in the timing that can be used for interfering with apoptosis initiation and prevention of cell damage. Moreover, due to the rAAV vector context, this technology could also be applicable for in vivo studies of a wide range of diseases characterized by caspase3-dependent cell death. In addition, a cell model of a constitutively active caspase-3 was developed. Chemical agents commonly used to induce apoptosis can act through a caspase-3-independent pathway and can fail to sufficiently activate caspase-3 in some cell lines. Our model circumvents these disadvantages of the chemical apoptosis models and would be particularly useful for the experiments when it is of high importance to ensure the presence of active caspase-3 in the cells.

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