Detection of caspase-3 activation in single cells by fluorescence resonance energy transfer during photodynamic therapy induced apoptosis
Introduction
Photodynamic therapy (PDT) is a new cancer treatment modality. Certain photosensitizing drugs can accumulate preferentially in lipophilic cell organelles, including mitochondria, endoplasmic reticulum, nucleus, and lysosomal membranes. The drug accumulated in the target tissue is activated by light of appropriate wavelengths [1], [2], [3]. Light activation of the drug induces formation of reactive oxygen species (ROS). The intermediates are capable to produce cellular damages that ultimately lead to cell death by either necrosis [4], [5], [6], [7], or, apoptosis [8], [9], [10], [11], [12], [13], [14], [15]. The production of ROS by PDT may also promote tissue destruction in part by vascular occlusion leading to ischemic necrosis [16].
Apoptosis is a very important cellular event that plays a key role in pathogeny and therapy of many diseases [17], [18]. The mechanisms of the initiation and regulation of apoptosis are complex and diverse [19]. In many apoptosis pathways, activation of the effector caspases is considered the final step [20]. Among the spectrum of various caspases, caspase-3 is believed to be the primary executioner of apoptosis. With inhibition of caspase-3 activation, cell apoptosis can be blocked [21]. When caspase-3 is activated, depending on the activation mechanism, it can induce chromatin condensation, DNA fragmentation, and cleavage of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) [22]. Because the caspases-3 activation is a landmark event in apoptosis, assaying caspases-3 has been widely used as a tool for detecting programmed cell death [23]. Caspase-3 activation is typically studied by western blotting or caspase-3 activation detection kits [24], [25]. Unfortunately, these techniques are time consuming and cannot be used to monitor the activation of caspases in real-time. Furthermore, the conventional detection techniques cannot be used to investigate the caspase-3 activation at single cell level.
The fluorescence resonance energy transfer (FRET) is a process by which transfer of energy occurs from a donor fluorophore molecule to an acceptor fluorophore molecule in close proximity. The emission spectrum of the donor molecule overlaps with the absorption spectrum of the acceptor molecule. When the two fluorophores are spatially close enough there is energy transfer between the donor and acceptor molecules. The excited donor transfers its energy to the acceptor. This results in a reduction in donor fluorescence emission and, at the same time, an increase in acceptor fluorescence emission [26]. FRET technique has been used widely to study protein–protein interaction in living cells [26], [27], [28]. Miura et al. [29] has constructed a FRET probe, SCAT3 that consists of a donor (enhanced cyan fluorescent protein, ECFP) and an acceptor (Venus, a mutant of yellow fluorescent protein). The linking sequence contains a caspase-3 cleavage, DEVD [30]. The activation of caspase-3 leads to the cleavage of the linker, thus, effectively reduces the FRET. Using FRET technique based on SCAT3, the spatial-temporal dynamics of caspase-3 activity in individual living cells can be monitored in real-time [29].
PDT induced caspase-3 activation has been reported by Grancille et al. [31] with conventional western blotting technique. In this work, we used SCAT3 as a FRET probe to study the caspase-3 activation in SCAT3-expressing human lung adenocarcinoma cell line (ASTC-a-1) during photosensitization-induced apoptosis. The objective of the study was to further the investigation of PDT induced caspase-3 activation in real-time, using FRET technique, within single cells.
Section snippets
Cell line
The human lung adenocarcinoma cell line (ASTC-a-1) was obtained from Department of Medicine, Jinan University and cultured in RM1640 supplemented with 10% fetal calf serum (FCS), penicillin (100 units/ml), and streptomycin (100 μg/ml) in 5% CO2, 95% air at 37 °C in humidified incubator.
For the FRET experiments, ASTC-a-1 cells were transfected with 1 μg plasmid DNA of SCAT3 and 10 μl of Lipofectin reagent (GIBCO) per 100 μl of serum-free medium at 37 °C for 8 h. The cells stably expressing SCAT3
Intracellular distribution and FRET efficiency of SCAT3
In order to detect the intracellular distribution of SCAT3 in the stably transfected ASTC-a-1 cells, the fluorescence of SCAT3 was collected by a confocal microscope. Venus fluorescence was observed in both cytoplasm and nucleus (Fig. 1). Venus fluorescence was more intense in nucleus than in cytoplasm, likely a result of higher expression of SCAT3 in nucleus. It has been reported that procaspase-3 localizes in the cytoplasm, caspase-3 activation is initiated firstly in the cytosol and the
Discussion
The present report demonstrates that FRET technique can be used monitor caspase-3 activation at single cell level in real-time. Caspase-3 is activated during PDT induced apoptosis that is confirmed by secondary techniques, i.e. Hoechst 33342 staining.
Photodynamic therapy employs a combination of a photosensitizing chemical and visible light to produce singlet oxygen and other ROS [30], [32], and these ROS can cause mitochondrial damage and induce apoptosis through release of cytochrome c and
Acknowledgements
We thank Dr Masayuki Miura of RIKEN Brain Science Institute for generously providing the SCAT3 used in the experiments; Ying Jin and Jinjun Wang for technical assistance.
This research is supported by the National Natural Science Foundation of China (60378043; 30470494), and the Natural Science Foundation of Guangdong Province (015012; 04010394)
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