Apoptosis - Enzyme Analysis
Studies on the nematode C. elegans showed that two genes (ced-3 and ced-4) were crucial to the process of apoptosis. The ced-4 geneproduct has homologues in mammalian cells, especially a family of cysteine proteases that are now known as caspases. There are a number of caspases in mammalian cells that have been shown to be involved in the early stages of apoptosis, e.g. Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9 and Caspase 10. The functions of these enzymes are not yet entirely clear, but it appears that after an initial signal to the cell to undergo apoptosis, they may be responsible for the activation, amplification and execution of the apoptotic cascade.
Because of the central importance of the caspases in apoptosis, their detection by flow cytometry has become widespread. We can detect the activity of enzymes implicated in apoptosis in three ways:- By detecting the active form of the enzyme using a specific antibody.
- By using a fluorochrome labelled peptide that binds to the active site of the enzyme.
- By using a non-fluorescent substrate for the enzyme which yields a fluorescent product if the enzyme is active (e.g. PhiPhiLux)
In this first example, HL60 cells have been treated with 1µM staurosporine for 4 hours. The cells have been fixed and permeabilised and probed with an antibody against the active form of caspase-3. Clearly a population of cells positive for the active form of the enzyme can be seen. This method allows simultaneous assessment of cell surface antigen (although not cell death) and because the cells may be fixed, it will allow a single assessment of multiple time points.
The second example uses a Fluorochrome-Labelled Inhibitor of Caspases (FLICA). These labelled peptides bind to the active form of the caspase. One such kit that we have used is the CaspaTag's kit from Intergen. This has a fluorescein-labelled peptide that will enter live cells. Following an hour's incubation, cells may be analysed with the addition of a dead cell discriminator such as Propidium Iodide, 7AAD or TO-PRO-3. In the example below, early apoptotic cells are seen as being FITC positive (i.e., these are cells that have active caspases) but PI negative (i.e., they are not yet classified as dead). Cells in each phase (live, apoptotic or dead) can easily be quantified.
The third method employs a well-established cytometric technique for the detection of active enzymes, where cells are loaded with a non-fluorogenic substrate for the enzyme in question which is cleaved to form a fluorescent product. OncoImmunin produce a product called PhiPhiLux; this can come in two forms - one with a green fluorescent product and the other with an orange fluorescent product (which would also be compatible with GFP positive cells). Here, MDCK cells have been induced by staurosporine - the cells that have cleaved the substrate show green fluorescence.
A major target of caspase 3 is Poly (ADP ribose) polymerase (PARP) which is cleaved during apoptosis and is often seen as one of its hallmarks. PARP cleavage can be detected by means of a specific antibody and a protocol for this is available here.
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