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

Abstract

Here we describe a novel method for imaging apoptosis in cells using a near-infrared fluorescent (NIRF) probe selective for caspase-1 (interleukin 1beta-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.

Single-cell imaging of retinal ganglion cell apoptosis with a cell-penetrating, activatable peptide probe in an in vivo glaucoma model

“…Previously, the targeting of peptide-based imaging agents has been limited to extracellular or cell-surface targets because of the barrier function of the cellular membrane. However, efficient delivery of imaging probes to the cell interior using cell-penetrating peptides has greatly expanded potential applications for molecular imaging (26)(27)(28)(29)(30)(31), similar to advances with cell-penetrating peptides in therapy (32)(33)(34)(35). Delivery of optically quenched, activatable, peptide-based imaging probes to the intracellular compartment makes selective retention and signal amplification possible, improving sensitivity and signal-to-noise ratios, as well as increasing the number of biochemical processes that can be assessed.…”

Noninvasive imaging of apoptosis in cardiovascular disease

“…Using a caspase-3 and -7 specific luciferase-based reagent, apoptosis was successfully imaged within human adenocarcinoma cells [55]. One approach employs a novel caspase-1 (ICE)-specific, activatable, near-infrared fluorescent probe as a means to image apoptosis in multiple models of apoptosis; this strategy was used to successfully image apoptosis noninvasively in mice [39]. In a combined diagnostic and therapeutic approach, a targeted photodynamic therapy agent with a caspase-3 activated fluorophore was able to induce and then detect apoptosis in tumors in vivo [56].…”

“…Imaging very low levels of apoptosis with targeted probes is challenging and may require the use of activatable imaging strategies that are chemically engineered to produce high levels of signal amplification in the presence of their molecular target, e.g. with caspaseactivatable molecular probes [4,39].…”

“…Imaging very low levels of apoptosis with targeted probes is challenging and may require the use of activatable imaging strategies that are chemically engineered to produce high levels of signal amplification in the presence of their molecular target, e.g. with caspaseactivatable molecular probes [4,39].Apoptosis however has been imaged in several models of heart failure and cardiomyopathy with higher constitutive levels of apoptosis. One such model utilizes mice genetically engineered to overexpress Gαq, a subunit of the cell-surface receptors involved in promoting cardiac myocyte hypertrophy (α1-adrenergic receptor, angiotensin II type 1 receptor, and endothelin-1 receptor) [75].…”

Molecular optical imaging: Applications leading to the development of present day therapeutics

“…In vivo experiments in mice bearing gliomas and injected with the HSV-ICE vector showed a specific activation of ICE near-infrared probe. 24 Thus, ICE-NIRF probe can be used in monitoring endogenous and vector-expressed caspase-1 activity in cells and should prove useful in monitoring endogenous and vector-expressed caspase-1 activity, and potentially apoptosis in cell culture and in vivo.…”