Apoptosis Detection In Living Tissues
The subject of cell death has recently become a focus of interest for researchers from a variety of diverse fields, including cell and molecular biology, oncology, immunology, embryology, endocrinology, hematology, and neurology. The interest expands to a wide range of molecular, biochemical, and morphological changes which not only pertain to the actual act of cell death, but also predispose the cell to respond to an environmental or intrinsic signal by death, regulate the initial steps leading to irreversible commitment to death, and activate the postmortem cell disposal machinery. Tissue homeostasis is maintained by a delicate balance between cell proliferation and cell death. Disturbance of this balance often leads to pathological cell accumulations for example in cancer. In this respect, much attention has been paid to the process of cell proliferation, while the phenomenon of cell death has long been overlooked. This has changed since it became evident that cell death may arise not only from externally inflicted mechanical or chemical damage, but also from a suicidal process that is controlled from within the cell. During the last decade, cell biology as well as oncology research has focused on this latter process of programmed cell death, referred to primarily as apoptosis. It is anticipated that understanding of the basic mechanisms that underlie apoptosis will offer potential new targets for therapeutic treatment of diseases. In order to conduct such research, techniques, and tools to reliably identify and enumerate death by apoptosis are essential.
Traditionally, assessment of apoptosis in tissue is based on microscopic methods, where sections for microscopy are taken from tissue invasively using biopsy or post mortem specimens. Clearly, these methods set limitations to many follow-up studies. More complex methods are needed to visualize apoptosis in living systems, laboratory animals or human bodies. Therefore, we aimed to establish an assay which permits to visualize and quantify apoptosis non-destructively in real time in living animal tumor tissues. However, admittingly, the usefulness of remodeling of the apoptosis mechanism in vivo is still an open discussion, since it is obvious that dying cells are removed from tissues of living mice through phagocytosis. It is also well known that the cells undergo lysis at the end of the apoptosis process and removal of cells can occur with different efficiencies in different tissues. Up to this endpoint of apoptosis, a sufficient time span is available during which apoptotic cells in tissue samples still can be detected. In addition, massive apoptosis can overwhelm the phagocytic potential of a tissue and virtually all apoptotic cells will then remain there for an extended period. Therefore, it should in principle be possible to develop an in vivo apoptosis detection and quantification method in live tumor tissues. One of the most important preconditions for the development of in vivo apoptosis detection assay is, whether this assay is capable to identify apoptotic changes at early stages.
Commonly used apoptosis detection methods are usually based on terminal morphological and biochemical events, such as specific changes in cell surface and nuclear morphology. Due to constantly expanding insights into the cell biology, detection of apoptotic cells now advancing to more specific methods, where early biochemical events associated with the execution and completion of apoptosis are central. Here, activation of intracellular proteases in living cells, especially of caspase-3, are among the key protagonists. The detection of activated caspase-3 is a valuable tool to identify dying cells before all the morphological features of apoptosis execution (for instance nuclear fragmentation) are present. Since no activation of the caspase cascade has been found in necrotic cell death, caspase activity should be specific marker for apoptosis detection. The ability of Caspase-3 to cleave various cellular substrates at specific residues is a central concept. Incorporation of specific caspase substrates into living cells and the detection of cleavage products (based on fluorescence resonance energy transfer-FRET) have been presented as novel assays for apoptosis detection. Several sensors based on the principles of fluorescence resonance energy transfer (FRET) between two fluorescent proteins, which are covalently linked together by a caspase recognition site that fulfil the preconditions of FRET have been described.
Xu at al. (1998) have developed EBFP-DEVD-EGFP construct, where the disruption of FRET upon caspase construct cleavage is identified by FACS analyses, thus allowing to distinguish activated caspase-3 in cells. Unfortunately, this and either similar assays, are applicable only for in vitro conditions. However, the concept itself should be transferable to the small animal tumor model. Therefore, a hybrid protein composed of a head-to-tail-linked tandem construct of Hc-red1 (t-HcRed1), a 14 amino acid linker containing the caspase-3 cleavage site DEVD and EGFP was generated by our research group. FRET between EGFP and t-HcRed1 in the intact sensor results in reduction of fluorescence lifetime of the donor – EGFP. This reduction can be correctly and reproducibly detected by fluorescence lifetime imaging microscopy - FLIM.
Activation of Caspase-3 in the process of apoptosis disrupts the covalent linkage between EGFP and tHcRed1 and effectively eliminates the FRET effect, thus conveying that a cell is apoptotic. Previously, the functionality of the sensor has been validated in vitro using different kinds of cancer cell lines and demonstrates specific and sensitive detection and quantification of apoptosis induced by anticancer drugs.
