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Neoplasia. 2004 Mar; 6(2): 95–105.
PMCID: PMC1502090

A Novel Method for Imaging Apoptosis Using a Caspase-1 Near-Infrared Fluorescent Probe1


Here we describe a novel method for imaging apoptosis in cells using a near-infrared fluorescent (NIRF) probe selective for caspase-1 (interleukin 1β-converting enzyme, ICE). This biocompatible, optically quenched ICE-NIRF probe incorporates a peptide substrate, which can be selectively cleaved by caspase-1, resulting in the release of fluorescence signal. The specificity of this probe for caspase-1 is supported by various lines of evidence: 1) activation by purified caspase-1, but not another caspase in vitro; 2) activation of the probe by infection of cells with a herpes simplex virus amplicon vector (HGC-ICE-lacZ) expressing a catalytically active caspase-1-lacZ fusion protein; 3) inhibition of HGC-ICE-lacZ vector-induced activation of the probe by coincubation with the caspase-1 inhibitor YVAD-cmk, but not with a caspase-3 inhibitor; and 4) activation of the probe following standard methods of inducing apoptosis with staurosporine, ganciclovir, or ionizing radiation in culture. These results indicate that this novel ICE-NIRF probe can be used in monitoring endogenous and vector-expressed caspase-1 activity in cells. Furthermore, tumor implant experiments indicate that this ICE-NIRF probe can be used to detect caspase-1 activity in living animals. This novel ICE-NIRF probe should prove useful in monitoring endogenous and vector-expressed caspase-1 activity, and potentially apoptosis in cell culture and in vivo.

Keywords: Caspase-1, near-infrared fluorescence (NIRF), apoptosis, brain tumors, HSV


The balance and coordination between cell proliferation and programmed death (apoptosis) is essential for normal physiology. Apoptosis is involved in embryonic development, immune cell processes, and tissue homeostasis. In addition, neurodegenerative disorders, acquired immunodeficiency syndrome, cancer, myelodysplastic syndromes, ischemia/reperfusion injury, and autoimmune disorders all involve inappropriate apoptosis [1]. The transformation of a normal cell to an apoptotic cell is typically characterized by loss of cell volume, plasma membrane blebbing, nuclear chromatin condensation and aggregation, and endonucleocytic degradation of DNA into nucleosomal fragments [2]. These cell changes occur following sequential activation of initiator and effector caspases [3–6]. Once activated, caspases, of which there are at least 14 different mammalian members [7–9], cleave a variety of structural, regulatory, and DNA repair proteins and, by doing so, disrupt important cellular processes and trigger cellular and morphological events associated with cell death [10]. Consequently, caspases are considered both initiators and executioners of apoptosis.

The role that caspases play in immune response and apoptosis is still being elucidated. Caspase-1, also known as interleukin 1β-converting enzyme (ICE), was originally characterized as cleaving inactive prointerleukin 1β to generate the active proinflammatory cytokine interleukin 1β and is considered an initiator caspase [11]. Studies suggest that the activation of caspase-1 is under the control of caspase-11 [12] and that caspase-11, in turn, is involved in caspase-3 activation and apoptosis induced by brain ischemia [13,14]. Caspase-1 is required for apoptosis in Rat-1 fibroblasts as well as other mammalian and insect cells [11,15–17]. Caspase-1 activation is also required for human prostate cancer cells to undergo apoptosis in response to transforming growth factor-β [18], and the majority of primary prostate cancer specimens (80%) have downregulation of caspase-1 expression, implicating this loss as a potential step in malignant progression [19]. Furthermore, studies have demonstrated the activation of caspase-1 in dying motor neurons in a mouse model of Cu2+,Zn2+ superoxide dismutase-mediated familial amyotrophic lateral sclerosis [20].

Visualization of cellular apoptosis has traditionally used several approaches, including labeling with annexin V [21] and terminal transferase-mediated dUTP nick end labeling (TUNEL) staining [22–24]. For both annexin V and TUNEL staining, cells or tissues must be removed from the animal and fixed, which may inadvertently cause cellular disruption. Examination of the specificity and sensitivity of TUNEL staining reveals a serious drawback in its inability to discriminate apoptotic from necrotic cells because necrotic cells also have free DNA ends after oxidative and toxic injury [25,26].

Technology to monitor apoptosis and activity of specific caspases in living cells is currently under development. Studies have shown that apoptosis can be noninvasively imaged in living cells in real time using a recombinant luciferase reporter molecule that has attenuated levels of activity when expressed in mammalian cells [27]. This reporter molecule is cleaved by caspase-3 in apoptotic cells, resulting in restoration of luciferase activity, which can be detected in living animals with bioluminescence imaging. In a complementary approach, we have investigated a method to image apoptosis noninvasively and dynamically over time using a near-infrared fluorescent (NIRF) probe activated by caspase-1. In previous studies, we have developed a series of novel activatable NIRF probes specific for various proteolytic enzymes, such as cathepsins [28,29], matrix metalloproteinases [30–33], and viral proteases [34]. This novel optical imaging technology has been used for detection of specific protease activities in vitro, in culture, and in vivo [35]. Here we examined the specificity of a newly developed ICENIRF imaging probe with purified enzymes and inhibitors in vitro. This probe was also used to monitor caspase-1 expression in association with apoptosis resulting from drugs, irradiation, or infection of cells with a herpes simplex virus (HSV) amplicon vector expressing caspase-1-lacZ in culture and from vector-infected tumor cells in vivo.

Materials and Methods

Generation of HSV Amplicons

The caspase-1-lacZ cassette (4.2 kb) was obtained from Dr. Jungying Yuan (Harvard Medical School, Boston, MA) [15] and cloned into the multiple cloning site (MCS) of the HSV amplicon plasmid, pHGCX [36], which contains an HSV origin of replication (oris), a cleavage/packaging signal (pac), and an expression cassette for enhanced green fluorescent protein (EGFP; Clontech, Palo Alto, CA) under an intermediate early viral promoter, IE4/5 (Figure 2A). The resulting amplicon, termed HGC-ICE-lacZ, was transformed into DH10B-competent cells and DNA from colonies was assessed by restriction digestions. The control amplicons were pHGCX and a version of pHGCX bearing coding sequences for firefly luciferase in the MCS, termed pHGC-Fluc [34].

Figure 2
HSV amplicon vector expressing caspase-1-lacZ induces apoptosis and activates ICE-NIRF probe. (A) pHGC-ICE-lacZ amplicon. ICE-lacZ cDNA was cloned into the multicloning site of the HSV amplicon pHGCX under the control of a CMV promoter. The pHGCX backbone ...

Cell Culture

The C6BVIK cell line expressing HSV-1 thymidine kinase (TK) was derived from the rat glioma line C6 [37] by Ezzeddine et al. [38]. The human glioma cell line Gli36 was originally obtained from Dr. Anthony Capagnoni (UCLA School of Medicine, Los Angeles, CA). 2-2 cells, Veroderived cells that constitutively express the HSV-1 ICP27 protein, were kindly provided by Dr. Rozanne Sandri-Goldin (UC Irvine, Irvine, CA) [39]. All cell lines were cultivated in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Carlsbad, CA) containing 100 U/ml penicillin, 100 µg/ml streptomycin (P/S), and 10% fetal bovine serum (FBS; D10P/S) with 500 µg/ml G418 (Geneticin; GIBCO, Carlsbad, CA).

Packaging of Amplicon DNA Into HSV-1 Virions and Titration of Vector Stocks

Vero 2-2 cells were cotransfected with pHGC-ICE-lacZ or pHGC-Fluc, fHSVΔpacΔ27 0+, and pEBH-ICP27 DNA using LipofectAMINE (GIBCO), as described [36]. After 3 days, the cells were scraped into the medium, the suspension was frozen and thawed three times, and cell debris was removed by centrifugation (10 minutes, 1400 x g). To concentrate the vector stocks, the supernatant was centrifuged for 2.5 hours at 100,000 x g through a 25% sucrose solution in phosphate-buffered saline (PBS), pH 7.4. The pellet was suspended in DMEM containing P/S, 10% FBS, and G418. To determine vector titers [transducing units (tu) per milliliter], 293 cells were infected and, 24 hours later, green fluorescent cells were counted under 488-nm excitation on a Zeiss microscope (Thornwood, NY).

Synthesis of Probe

The NIRF probe was prepared following a previously published protocol [28]. Briefly, a caspase-1-cleavable peptide substrate [Gly-Trp-Glu-His-Asp-Gly-Lys fluorescein isothiocyanate (FITC)-Cys-NH2] [40] that contains an N-terminal NIR fluorochrome, Cy5.5 (absorption maximum 675 nm, emission maximum 694 nm; Amersham-Pharmacia, Piscataway, NJ), was attached to a biocompatible, partially pegylated poly-l-lysine delivery vehicle through the C-terminal cysteine residue to generate the ICE-NIRF probe (Figure 1A). On average, each delivery molecule contained 18 reporter probes with efficient autoquenching of fluorescence.

Figure 1
Characteristics of ICE-NIRF probe. (A) Schematic diagram of the ICE-NIRF probe. In its intact state (left), the high local density of fluorochromes causes substantial fluorescence quenching. The enzymatic activation by caspase-1, involving cleavage of ...

In Vitro Activation with Purified Caspases

Specific activation of the ICE-NIRF probe was tested in the presence of purified caspase-1 or caspase-3 in PBS buffer. The probe (0.2 µM, 500 µl) was incubated with or without specific caspase (0.25 µg; Calbiochem, San Diego, CA) at room temperature for 30 minutes. NIRF signal was measured using a fluorescence spectrophotometer (U4500; Hitachi, Chula Vista, CA). The excitation and emission wavelengths were set at 675 and 694 nm, respectively. In an inhibition experiment, a caspase-1 specific inhibitor, Ac-Tyr-Val-Ala-Asp-CHO (4 µM; Calbiochem), was added to the caspase-1 ICE-NIRF probe mixture and studied under the same conditions.

Assessment of Apoptosis by NIRF Probe and Annexin, Propidium Iodide (PI), and TUNEL Staining

First, cells were treated with 1 µM ICE-NIRF probe diluted in OptiMEM and incubated for 30 minutes in a 5% CO2 incubator. Then cells were rinsed twice with PBS and resuspended in 1 x binding buffer (Apoptosis Kit; Clontech) with 0.5 µg/ml annexin V-FITC and, in some cases, 1.25 µg/ml PI per well using the annexin V-FITC Apoptosis Kit (Clontech) and incubated for 5 to 15 minutes in the dark. Following rinsing with OptiMEM, cells were fixed with 2% paraformaldehyde (PFA) for 15 minutes, washed with PBS twice, and mounted using Fluoromount G media (Southern Biotechnology, Birmingham, AL). The slides were observed under the confocal microscope (Zeiss Axiovert 200) using the 633-nm laser to detect the NIRF probe, the 488-nm laser to detect EGFP and annexin, and the 543-nm laser to detect PI staining. Assessment of apoptosis was also performed using TUNEL staining [41] and detected with the 543-nm laser. The LSM 5 Pascal Software (v. 2.8 WS) was used for quantitative analysis of images. The “Profile” function of this software package was used to compute the intensity as a function of distance along a line drawn directly through each image. The intensities were averaged, and the average with the 63-nm laser was normalized to the average with the 488-nm laser.

Drug Treatment

In order to evaluate the specificity of the ICE-NIRF probe, we examined the effects of the caspase-1 inhibitor, YVAD-cmk (Clontech), and the caspase-3 inhibitor, DEVD-fmk (Clontech), on HGC-ICE-lacZ vector-induced probe activation. For these experiments, Gli36 cells were plated at a density of 3 x 105 cells/well in 24-well plates (Becton Dickinson, Franklin Lakes, NJ), and infected 24 hours later with either HGC-ICE-lacZ or HGC-Fluc vector. Vectors were used at a multiplicity of infection (MOI) = 1 tu/cell, at which over 90% of cells were infected as assessed by EGFP fluorescence. For the caspase-1 inhibitor, 24 hours after being plated, cells were coincubated with 25 µM YVAD-cmk and HGC-ICE-lacZ vector (MOI = 1) for 24 hours. For the caspase-3 inhibitor, a parallel experiment was carried out using 10 µM DEVD-fmk. To evaluate caspase-1 activity and apoptosis, cells were assessed for NIRF probe activation and annexin and TUNEL staining, as above. Tetrazolium salt WST-1 cell viability assays were performed to confirm that these inhibitors were not toxic to Gli36 cells under these conditions.

To induce apoptosis, Gli36 cells were exposed to 50 µM staurosporine (Sigma, St. Louis, MO) for 24 hours, then washed twice in PBS and evaluated for ICE-NIRF probe fluorescence and annexin staining. Control cells were exposed to 0.01% DMSO, the same percentage as in the staurosporine-treated cells.

Based on cell viability experiments, 100 µg/ml ganciclovir (Cytovene-IV; Roche Laboratories, Nutley, NJ), which gave 69% viability of C6VIK cells after 7 days of exposure, was used for culture experiments. C6VIK cells were plated at 1 x 105 per well in a 24-well plate. Twenty-four hours after plating, ganciclovir (100 µg/ml) was applied for time points ranging from 1 to 7 days. Fresh ganciclovir was added to media every other day. At each time point, media and drug were removed, cells washed twice with PBS, ICE-NIRF probe, and annexin were applied, and then TUNEL staining was carried out and the slides were evaluated 24 hours later by confocal microscopy.

Cell Viability Assays

Viability was assessed with the cell proliferation reagent, WST-1 (Roche Molecular Biochemicals, Penzberg, Germany), for Gli36 cells treated with the caspase-3 inhibitor and for C6VIK cells treated with ganciclovir. One thousand cells per well were plated in a 96-well plastic plate and, 24 hours later, the drug was added at the following concentrations: 5, 10, and 25 µM for DEVD-fmk and 50, 100, and 150 µg/ml for ganciclovir. Cells were exposed to the drug for 24 hours or 7 days, respectively, at which time media was aspirated and cells were incubated in 100 µl of fresh media and 10 µl of WST-1 reagent for 4 hours at 37°C in a 5% CO2 incubator. Readings were taken on a Spectra max Plus 384 spectrophotometer (Molecular Devices Corporation) at 420 nm wavelength, and data were analyzed using Prism 3.03. Drug concentrations were tested in triplicate.

Granule Cell Preparation

Cultures enriched in granule neurons were prepared from dissociated cerebella of 5-day-old mice (129Sv/C57B6 mixed background), as described [42]. One day after culturing, the antimitotic drug, cytosine-d-arabinofuranoside (10 µM), was added to prevent proliferation of non-neuronal cells. After 3 days in culture, cells were irradiated with 14 Gy delivered from a 137Cs source at 5.37 Gy/min. Twenty-four hours after irradiation, cells were incubated with the NIRF probe and annexin, as described above.

In Vivo NIRF Imaging

In an experimental in vivo implant model, Gli36 cells were infected in culture with HGCX-ICE-lacZ or HGCX vector at an MOI = 1.0. Four hours postinfection, cells were trypsinized, collected by centrifugation at 3000 x g for 5 minutes, and washed twice in PBS; and the 4 x 106 cells resuspended in 50 µl of PBS, mixed with 50 µl growth media, and then injected subcutaneously bilaterally under the forearms of five nude mice. Anesthesized mice were imaged 24 hours following intravenous (i.v.) probe administration of the ICE-NIRF probe (2 nmol of ICE-NIRF probe per animal) using a whole-body NIRF imaging system (CMIR, Charlestown, MA).

The imaging system consisted of a light-tight box equipped with a 150-W halogen lamp and an excitation filter system for Cy5.5 (610–650 nm; Omega Optical, Brattlerburo, VT). The field of view was homogenous to ± 10% over a 5 x 5-cm area through the use of fiberoptic cables and beam diffusers. Fluorescence was detected by a 12-bit monochrome CCD camera (Photometrics, Tuscon, AZ; Kodak, Rochester, NY) equipped with a C-mount lens and an emission filter at 700 nm (Omega Optical). Images were digitally acquired as 16-bit Tiff files and processed on a Macintosh computer using commercially available software (IP Lab Spectrum; Signal Analytics, Vienna, VA) [29]. Brightfield and NIRF images were collected and images superimposed. Quantification of fluorescence was done using CMIR image program by finding the mean pixel intensity of each implant infected with HGC-ICE or HGCX, as well as the background fluorescence from an adjacent region in each mouse.

Statistical Analysis

Statistical analysis was conducted using Microsoft Excel 97 SR-2. For cell culture experiments, a two-sample t-test assuming equal variances was conducted by comparing the average intensity of the probe signal across the microscopic field at 633 nm normalized to the average intensity of the EGFP signal at 488 nm between groups. For in vivo experiments, statistical analysis comparing the mean pixel intensities between treatment groups was conducted using a t-test: paired two sample for means, which gave a P (T < -t) one-tail value.


Specificity of NIRF Probe for Caspase-1

The specificity of the ICE-NIRF probe for caspase-1 (Figure 1A) was evaluated in two ways: 1) incubation of the probe with purified caspase-1 or caspase-3 in vitro; and 2) incubation of HGC-ICE-lacZ vector-infected cells in culture with the probe in the presence of a caspase-1 inhibitor or a caspase-3 inhibitor. For in vitro testing, the probe was incubated with or without purified caspase-1 or caspase-3 over 30 minutes at room temperature. Incubation of the probe with purified caspase-1 resulted in a 78-fold increase in fluorescence (Figure 1B). Activation of the probe by caspase-1 was blocked over 95% by coincubation with a caspase-1-specific inhibitor. In comparison, the incubation of the probe with purified caspase-3 caused no changes in fluorescence signal.

In order to deliver the caspase-1-lacZ fusion protein to cells, coding sequences were placed under the CMV promoter in an amplicon plasmid, pHGCX [35], yielding pHGC-ICE-lacZ (Figure 2A). For convenient monitoring of gene expression, this construct includes a reporter EGFP cassette under an immediate early viral promoter (IE4/5). A parallel amplicon construct containing luciferase under the CMV promoter, pHGC-Fluc, was used as a control vector.

Gli36 cells were infected with packaged HGC-ICE-lacZ or control vector at an MOI = 1. Twenty-four hours following infection, cells evaluated for ICE-NIRF probe fluorescence indicated a specific signal in HGC-ICE-lacZ vector-infected cells (Figure 2D). Infection of Gli36 cells with the HGC-ICE-lacZ vector also induced apoptosis indicated by positive TUNEL staining (Figure 2C) concomitant with activation of the ICE-NIRF probe. Both apoptosis and probe activation were substantially blocked by incubation of cells infected with the HGC-ICE-lacZ vector with the caspase-1 inhibitor, YVAD-cmk, indicating specificity for caspase-1 (Figure 2, F and G). Infection of Gli36 cells with the control vector, HGC-Fluc, did not induce apoptosis, indicated by the absence of TUNEL staining, nor did it produce a NIRF fluorescence signal (data not shown).

The effect of caspase-1 and caspase-3 inhibitors on HGC-ICE-lacZ-induced activation of the ICE-NIRF probe by Gli36 cells was quantified by determining the average intensity of the probe signal across the microscopic field at 633 nm and normalizing that to the average intensity of the EGFP signal at 488 nm (Figure 2H). The caspase-1 inhibitor prevented HGC-ICE-lacZ vector-mediated activation of the probe. A two-sample t-test assuming equal variances indicated that this difference was significant (P one-tail ≤ .0157, n = 3). However, there was no significant difference in NIRF probe activation by HGC-ICE-lacZ in the absence or presence of the caspase-3 inhibitor (P ≤ .166), and the control vector did not activate the probe.

ICE-NIRF Probe Detects Apoptosis in Cultured Cells

Several standard methods for inducing apoptosis were used to determine whether this ICE-NIRF probe could be used to monitor caspase-1 activity during externally elicited apoptosis in living cells in culture. One such method for inducing apoptosis involves treating cells with staurosporine [43], a microbial alkaloid, which inhibits protein kinases [44]. Treatment of Gli36 glioma cells with 50 µM staurosporine for 24 hours induced apoptosis, as assessed by positive annexin staining (Figure 3A), and also activated the ICE-NIRF probe (Figure 3B). Control cells treated with 0.01% DMSO, the same percentage as in the experimental treatment, did not show significant annexin staining (Figure 3D) or an ICE-NIRF signal (Figure 3E). In order to examine the specific role of caspase-1 in staurosporine-induced apoptosis and probe activation, cells were coincubated with both the caspase-1 inhibitor and staurosporine prior to treatment with probe and annexin (Figure 3G–I). The caspase-1 inhibitor decreased probe activation (Figure 3H), but did not completely block apoptosis (Figure 3G). Thus, a single caspase inhibitor for caspase-1 was not enough to save the cells from apoptosis triggered by staurosporine, but did block activation of the ICE-NIRF probe. Quantification of fluorescence intensity indicated that, overall, the caspase-1 inhibitor reduced staurosporine-induced ICE-NIRF activation by 80%.

Figure 3
Induction of apoptosis with staurosporine resulting in activation of the ICE-NIRF probe. Gli36 cells were treated with 50 µM staurosporine for 24 hours (A–C) or with the same percentage of DMSO (0.01%) to which experimental wells were ...

Prior studies have demonstrated that C6BVIK glioma cells stably expressing HSV-TK die in response to treatment with ganciclovir [38] and that ganciclovir kills by apoptosis [45]. Viral TK is a prodrug-activating enzyme, which converts ganciclovir to a toxic nucleoside analogue. First, C6BVIK cells were treated with concentrations of ganciclovir ranging from 50 to 150 µg/ml for 7 days and assayed for cell viability using a WST assay. All concentrations resulted in significant cell death as compared to their respective controls (P ≤ .01) with about 40% loss of viability at 150 µg/ml ganciclovir (Figure 4A). C6BVIK cells treated with ganciclovir for 1 to 7 days were also assessed for apoptosis and probe activation by staining with annexin and the ICE-NIRF probe. For time points 3 to 7 days in the presence of ganciclovir (100 µg/ml), condensed apoptotic nuclei were visible with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI) or PI staining (data not shown). C6BVIK cells treated with ganciclovir at 100 µg/ml for 3 days yielded positive annexin staining (Figure 4B) and ICE-NIRF probe activation (Figure 4C). Untreated C6BVIK cells did not stain positively for annexin (Figure 4E) or demonstrate ICE-NIRF fluorescence (Figure 4F).

Figure 4
Treatment of C6BVIK cells expressing HSV-TK with ganciclovir induces apoptosis and activates caspase-1 NIRF probe. (A) C6BVIK cells were treated with 50 to 150 µg/ml ganciclovir and assayed for cell viability using a WST assay. Concentrations ...

We also examined whether induction of apoptosis by irradiation of primary neuronal cultures of cerebellar granule cells would lead to activation of the ICE-NIRF probe. Previous studies have shown that irradiation of the cerebellum of newborn mice results in extensive apoptosis in the external granule layer [46]. Cultured granule cells from newborn mice were irradiated and, 24 hours later, cultures were incubated with ICE-NIRF probe and stained with annexin. Positive annexin staining was observed in irradiated granular cells (Figure 5A) in parallel with activation of the ICE-NIRF probe (Figure 5B). Minimal cellular annexin staining and no detectable ICE-NIRF activation were observed in nonirradiated granule cells (Figure 5, D and E, respectively).

Figure 5
Induction of apoptosis by irradiation of cerebellar granule cells activates ICE-NIRF probe. Twenty-four hours following irradiation, cultured cells were incubated with probe and stained with annexin. Positive annexin staining (A) and activation of the ...

In Vivo Imaging

In an experimental in vivo tumor implant model, Gli36 cells were infected in culture with HGC-ICE-lacZ or HGCX virus vectors at an MOI = 1 for 4 hours, which yielded > 95% infection of cells. Infected cells were implanted bilaterally (4 x 106 per site) in the forearms of five nude mice. Twenty-four hours later, the ICE-NIRF probe was injected i.v. and the mice imaged 24 hours after probe administration. The mean pixel intensity of the HGC-ICE-lacZ-infected Gli36 implant was 769.3 AU (SD + 209.2), whereas that of the HGCX-infected implant was 630.6 AU (SD + 101.0), and the background in an adjacent area was 436.3 (SD + 77.7). This background area had low endogenous fluorescence, whereas nonspecific probe fluorescence was somewhat higher in the liver, spleen, and bladder, as seen for other NIRF probes [28]. Subtracting the adjacent background from the implant values, there was significantly (P ≤ .006) more fluorescence in HGC-ICE-lacZ-infected cells by 76% than in HGCX-infected cells. A pseudocolor image of the mean pixel intensities of NIRF fluorescence minus the background in implants in one of these mice indicates that this increase in intensity is visually notable (Figure 6). Of note, the NIRF fluorescence of the HGCX-infected cell implant was significantly (P ≤ .048) higher than the adjacent normal tissue, suggesting that the vector or injectate volume may have triggered tissue damage and apoptosis. A direct comparison of the fluorescence between the HGC-ICE-lacZ-infected cell implant and adjacent normal tissues indicated a 1.7-fold increase in mean intensity.

Figure 6
Pseudocolor image of caspase-1 activity in vivo. HGC-ICE-lacZ-infected Gli36 cells were implanted into the left side of a nude mouse and HGCX-infected Gli36 cells into the right side. Animals were imaged 48 hours after injection of cells, 24 hours after ...


Here, we describe the development and characterization of a caspase-1 (ICE)-specific NIRF probe as a means to noninvasively image apoptosis in living cells. Specificity was shown by selective probe activation with caspase-1, and not caspase-3, and inhibition of activation by a caspase-1 inhibitor, and not a caspase-3 inhibitor. Furthermore, induction of apoptosis and probe activation was demonstrated following delivery of a caspase-1-lacZ fusion protein to cells in culture using an HSV amplicon vector. Three different methods of inducing apoptosis—staurosporine treatment, exposure of cells expressing HSV-TK to ganciclovir, and irradiation of cerebellar granule cells—all led to activation of this ICE-NIRF probe. The caspase-1 inhibitor partially blocked staurosporine-induced activation of the probe, but did not completely block apoptosis, suggesting that staurosporine induces apoptosis by activating multiple caspase pathways, some independent of caspase-1, such as activation of the CED-3 family of proteases [47].

Caspase-1 has been implicated in apoptosis of a number of cell types. Caspase-1 activity is present in cells throughout the lifespan of mice and may be an early mediator of cell death during some types of neuronal degeneration, with caspase-1 acting as a chronic initiator and caspase-3 acting as the final executioner of cell death. Caspase-1-deficient mice show delayed neutrophil apoptosis and a prolonged inflammatory response to lipopolysaccharide-induced acute lung injury, but otherwise normal development and physiology [48]. Caspase-1 is activated as an early event in the death of motor neurons in transgenic mouse lines expressing a mutant form of Cu2+,Zn2+ superoxide dismutase, which develop familial amyotrophic lateral sclerosis-like disease [20]. In these animals, activation of caspase-1 is followed by activation of caspase-3 and the appearance of apoptotic neurons and astrocytes specifically in affected regions.

Our laboratory has previously expressed the ICE-lacZ fusion protein in experimental cancer therapy using a tetracycline-regulated retrovirus vector [49]. In the case of slow-growing, benign tumors, where reduction in volume can be therapeutic and the rate of DNA replication may not make cells susceptible to DNA-damaging agents, delivery of apoptotic proteins, such as ICE-lacZ, may prove efficacious. Targeting could be achieved by direct injection of vectors into the tumor mass with consequent volume reduction. In this context, NIRF imaging could allow direct monitoring of therapeutic gene expression.

Currently, most ways of studying apoptosis rely on experiments done on cell extracts in the test tube or imaging of cells in culture or tissue sections. The former includes DNA fragmentation or fluorogenic assays using fluorescently labeled substrates for various caspases, and the latter includes surface labeling of annexin V, nuclear staining with PI or TUNEL, or assessment of caspase activities in intact cells using cell-permeable fluorogenic caspase substrates. Recently, a fluorescent Cy5.5-annexin V-NIRF probe has shown promise in imaging apoptosis in living animals [50]. The key issue in converting annexin V from a cellular imaging agent to an in vivo imaging agent was the use of a fluorochrome, which emits fluorescence light in the near-infrared range. Light in the NIRF range allows much deeper tissue penetration, as well as less nonspecific tissue autofluorescence and reflectance [51].

The potential for imaging of caspase-1 activity in vivo with the ICE-NIRF probe was evaluated in an experimental tumor implantation model. Glioma cells were infected in culture with an HSV amplicon vector encoding caspase-1-lacZ (HGC-ICE-lacZ) or a control vector, implanted in mice, and evaluated 48 hours later after i.v. ICE-NIRF probe injection. There was a significant increase (76%) in fluorescence in the ICE-lacZ vector-infected cells, with a greater increase (171%) between ICE-lacZ vector-infected cells and adjacent normal tissue background. The low—but greater than background—levels of ICE-NIRF fluorescence observed in the implants infected with control amplicon vector may be due to apoptosis induced by the vector or injectate. These in vivo data suggest that this ICE-NIRF probe has the potential to image caspase-1 activity and apoptosis, depending on enzyme levels and tissue depth.

Compared to other methods, imaging apoptosis with an ICE-NIRF probe has the potential advantage of allowing much deeper tissue penetration, with less nonspecific tissue autofluorescence, as compared to imaging using a recombinant luciferase reporter molecule and bioluminescence imaging, as photons do not allow deep tissue penetration [27]. Furthermore, given the numerous types of caspases with roles in different biologic processes, it will be highly advantageous to have probes for in vivo imaging that are specific to the different caspases. Over the past few years, we have applied several NIRF protease activatable probes to various biological systems and have shown great promise of imaging disease processes through their associated protease activity [35,52]. The applications include cancer, arteriosclerosis, arthritis, blood coagulation, inflammation, and others. In conclusion, this novel ICE-NIRF imaging probe allows high-sensitivity imaging of apoptosis involving caspase-1 activation in living cells. It should have important applications in imaging results of therapeutic intervention in cancer, extent of cell damage in injury models, and normal apoptotic mechanisms in development.


We thank Suzanne McDavitt for skilled editorial assistance and Dr. Jungying Yuan (Harvard Medical School) for providing the plasmid pBSMIOZ containing the caspase-1-lacZ cassette.


1This work was funded by the MGH Medical Discovery award (S.M.M.), the Department of the Army DAMD17-00-1-0537, the Texas Neurofibromatosis Foundation through the Terrill family (X.O.B.), NIH P50-CA86355, R24-CA92782 (R.W.), and R33-CA88365 (C.H.T.).


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