Apoptosis was originally defined by the morphological changes that occur with remarkable fidelity in a wide variety of cell types, regardless of cell lineage and the method of cell death induction. The morphological changes can occur rapidly after induction and include cell surface blebbing, cell and nuclear shrinkage. Consideration of morphology is the single most important method for detection of apoptosis and no other biochemical test or assay is available to replace microscopic observation. Researchers require alternative methods for scoring apoptosis that provide supportive biochemical evidence or quantitation of data. This can be particularly important in vivo when the later, more obvious morphological changes may not occur due to phagocytosis and cell removal, and in experimental systems when large sample numbers have to be assayed and screened. Once morphological evidence has determined that the mechanism of cell death occurs by apoptosis, continued stringent analysis using morphology is not necessary and a more convenient biochemical method is more appropriate. As elucidation of the complex and varied biochemical pathways leading to the execution of apoptosis continues, it is likely that the options available for apoptosis detection will continue to expand.
Cell Membrane Events
Some of the earliest apoptotic changes occur at the cell surface. The early recognition of apoptotic cells by phagocytic cells, the significant loss of water leading to cell shrinkage, and the maintenance of intact cell membranes, despite the cell surface blebbing observed in many cell types, indicate significant changes have occurred in the plasma membrane. One of the better understood cell surface modifications is exposure of phosphatidylserine (PS). Normally, PS is confined to the inner layer of the lipid bilayer. This asymmetric distribution is maintained in normal cells by the action of specific proteins. After induction of apoptosis under the influence of translocases, the PS is flipped from the inner to the outer bilayer, rendering the molecule available for detection. Annexin V is a blood clotting factor that exhibits a high calcium-dependent specificity for PS binding. The coupling of annexin to fluorescein, or biotin, generates a direct, rapid, and simple method for the detection of apoptosis on unfixed cells.
Although organelles remain grossly normal during the earlier stages of apoptosis, research has revealed a pivotal role for mitochondria in the apoptotic cascade. The energy generated during cellular respiration accumulates in the mitochondrial transmembrane space as an electron gradient called the mitochondrial membrane potential or Δψm. This electrochemical gradient is often disturbed during apoptosis and can be detected using cationic dyes such as DePsipher™ (5,5’6,6’- tetrachloro-1,1’,3,3’-tetraethylbenz-imidazolylcarbocyanine iodide) or MitoShift™ (tetramethylrhodamine ethyl ester). DePsipher™ readily enters cells and accumulates as a multimeric aggregate within healthy mitochondria, and under UV light, this multimeric form emits red light. In apoptotic cells, the mitochondrial membrane potential collapses and the dye returns to a monomeric form. The monomeric molecule emits green fluorescence under UV light. DePsipher™ provides a rapid fluorescence-based assay for the apoptosis-associated loss of mitochondrial potential.
Activation of caspases, or cysteinyl proteases, is a necessary event for execution of the apoptotic response. Some of the caspases are activated early in the apoptotic process and their activation is the first step in a cascade of proteolytic cleavage of key proteins and enzymes, including other caspases and poly (ADP-ribose) polymerase (PARP). The caspases each cleave a defined amino acid sequence. This specificity has led to the development of highly specific irreversible peptide inhibitors. Delivery of these peptides allows for complete inhibition in the execution of apoptosis, thereby providing a means to probe the early events in apoptosis and to investigate the ordering of key events in the process. Since the substrate specificity of the caspases is high, analysis of substrate cleavage also provides a useful biochemical marker.
The Bcl-2 family of proteins is well known to be important in determining cell fate, although their mechanism of action remains unclear. The relative levels of the family members, as well as the subcellular distribution of these proteins, changes during apoptosis. In particular, the movement of some members from the cytoplasm to the mitochondria and the subsequent associations that occur between the Bcl-2 family members and other mitochondrial membrane associated proteins are indicated to be crucial steps in apoptosis. Antibodies that recognize the individual Bcl-2 family members provide powerful tools for studying alterations in expression levels, the subcellular members provide powerful tools for studying alterations in expression levels, the subcellular redistribution, and protein associations of these intriguing molecules.
DNA fragmentation occurs as one of the final stages of cell death and has long been considered a hallmark of apoptosis and one of the defining biochemical events of the pathway. DNA fragments are generated initially through single-stranded breaks that produce fragments of DNA larger than 50,000 bases. Later in the process, double-stranded DNA cleavage occurs mainly in the linker regions between nucleosomes. The length of DNA wrapped around the histone core within nucleosomes is approximately 200 bases. The cleavage generates DNA ends with a free 3’ hydroxyl group. For detection of double-strand DNA breaks, anti-γH2AX is unique in only detecting phosphorylated histones at sites of double-strand DNA breaks. Trevigen’s phosphorylated γH2AX antibody has been qualified for both Western blotting and immuno-histochemistry.
For detection of the DNA fragmentation associated with apoptosis by DNA laddering, the DNA is isolated and the cleaved fragments are separated by agarose gel electrophoresis. If sufficient DNA is present, staining of the gel with ethidium bromide reveals the typical laddering pattern of multimers of 180–220 bases. Trevigen’s Ethidium Bromide DNA Laddering kit provides the necessary reagents for detection of the DNA ladder.
DNA fragmentation can also be detected within the nuclei of cells and tissues. Many of the standard fixatives used for preserving cells and tissues maintain the integrity of the DNA and the free 3’ hydroxyl groups at the sites of cleavage. The DNA thus provides a target for terminal deoxynucleotidyl transferase (TdT), which can sequentially add nucleotides to the 3’ end of DNA. The addition of a nucleotide tagged with bromine or biotin in the reaction mix then allows indirect visualization of any DNA labeling by TdT within fixed cells or tissue sections. Utilizing a TUNEL-based assay, Trevigen has developed a series of kits for the in situ detection of apoptosis with colorimetric and fluorometric options. The TACS® kits are tailored for the detection of DNA fragmentation associated with apoptosis in a variety of cell and tissue types and for analysis by different formats that include microscopy, flow cytometry, and 96 well plates.
*TACS: Trevigen Apoptotic Cell System*