What is the progression of events in apoptosis and what Promega products are available for its study?

Apoptosis can be triggered by a variety of stimuli. These stimuli can be  split into two broad groups: i) receptor-mediated and ii) chemical-mediated (1). The classic example of receptor-mediated apoptosis is Fas Ligand (or anti-Fas antibody) binding to the Fas (CD95/Apo-1) receptor. This results in trimerization of the Fas receptor and recruitment to the intracellular domain of the receptor or adaptor proteins such as FADD/MORT1 (Fas-Associated Death Domain) and TRADD (TNF Receptor-Associated Death Domain). In turn, the death effector domain (DED) of these adaptors interacts with the corresponding DED regions in the N-terminus of procaspase-8. This results in the cleavage of procaspase-8 to yield active caspase-8. Genetic evidence showing that caspase-8 is required for Fas-induced apoptosis, together with in vitro evidence that it can activate all other known caspases, implicate this as the initiator caspase for receptor mediated apoptosis (2,3). The active caspase-8 cleaves and activates the so-called effector caspases (such as caspase-3 and -7) in the caspase cascade. In addition, active caspase-8 can cleave Bid (a pro-apoptotic Bcl-2 family member), which results in translocation of the cleaved Bid to the mitochondria and subsequent release of cytochrome C (4,5). The released cytochrome C forms a complex with Apaf-1 in the presence of dATP. This complex then recruits and cleaves procaspase-9 to yield activated caspase-9 (6). The active caspase-9 can then act together with caspase-8 to directly activate the effector caspases.

In chemical-mediated apoptosis, cytochrome C release from mitochondria occurs prior to caspase activation. Presumably this release is caused by a mechanism that is independent of Bid cleavage, such as  an interaction of Bid with the pro-apoptotic protein Bax (7). Following release of cytochrome C, caspase-9 is activated in much the same way as described above for receptor-mediated apoptosis.

In both cases, the late apoptotic events take place after activation of the effector caspases. These events include exposure of phosphatidylserine on the external surface of the plasma membrane (Annexin V binding), cleavage of Poly (ADP-ribose) Polymerase (PARP) and internucleosomal DNA fragmentation.

Promega has several products available for the study of both the early and late stages of apoptosis. The CaspACE^TM Assay System, Colorimetric (Cat.# G7351, G7220), and the CaspACE^TM Assay System, Fluorometric (Cat.# G3540), can be used to assay for caspase-1 or caspase-3 activity in apoptotic cells. In addition, the new Anti-ACTIVE^® Caspase-3 pAb (Cat.# G7481) can be used for immunocytochemical detection of active caspase-3 in apoptotic cells. For direct labeling of apoptotic cells, the CaspACE^TM FITC-VAD-FMK In Situ Marker (Cat.# G7461, G7462) can be used. This product is a cell-permeable fluoroscein isothiocyanate (FITC) conjugate of the pan-caspase inhibitor VAD-FMK, which irreversibly binds to activated caspases, thereby specifically labeling apoptotic cells in situ. Similarly, the Caspase Inhibitor Z-VAD-FMK (Cat.# G7231, G7232), a cell-permeable pan-caspase inhibitor, is available to study caspase-dependent and-independent pathways in apoptosis.

For the study of cytochrome C localization, Promega supplies Anti-Cytochrome C mAb (Cat.# G7421), a mouse monoclonal antibody against cytochrome C. Generally, a double staining procedure is performed using a mitochondrial-specific dye such as CMX-rosamine. In nonapoptotic cells, the cytochrome C labeling should give a punctate staining that mirrors that of the CMX-rosamine. In apoptotic cells, cytochrome C is released, and this colocalization of staining disappears. In most cases it may not be possible to see any staining at all, as cytochrome C becomes unstable once it is released into the cytoplasm. Therefore, it is important to have a nonapoptotic control to ensure that the staining conditions used are able to detect any available cytochrome C.

For the late stages of apoptosis,  the p85 caspase cleavage product of PARP can be identified using Promega's Anti-PARP p85 Fragment pAb* (Cat.# G7341). This product is designed for immunocytochemical staining and specifically recognizes the cleaved form of PARP. It does not recognize the intact full-length PARP protein. Thus, positive staining with this antibody is an indication that the cells are apoptotic. Finally, Promega also offers two TUNEL assay kits used to assay for DNA fragmentation, a hallmark of apoptosis. The Apoptosis Detection System, Fluorescein (Cat.# G3250) uses terminal deoxynucleotidyl transferase (Tdt) to add fluorescein-12-dUTP* to the 3´-OH ends of fragmented DNA. This labeling can then be visualized using a fluorescence microscope or by Fluorescent Activated Cell Sorting (FACS^TM) analysis. For researchers who prefer a colorimetric assay, the DeadEnd™ Colorimetric Apoptosis Detection System (Cat.# G7130, G7360) labels the 3´-OH ends of fragmented DNA with a biotinylated nucleotide. The biotinylated fragment can subsequently be detected with a streptavidin-HRP conjugate and a precipitable HRP substrate (DAB and hydrogen peroxide). The apoptotic nuclei can then be visualized under a standard light microscope and appear brown.

The CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (Cat.# G3580, Cat.# G3581, Cat.# G3582) provides in vitro information about cell viability by measuring the capacity of cells to reduce MTS, a tetrazolium compound, into a colored formazan product. Active cell metabolism results in the generation of reducing equivalents such as NADH and is necessary for tetrazolium reduction. An increase in the conversion of MTS to the colored formazan product provides a relative measure of viable cell number, while a decrease provides a relative measure of cell death.

Recently, it has been shown that the first death effector domain (DED) of caspase-8 can activate the c-Jun N-terminal kinase (JNK) pathway independent of its caspase activity and that a mutation in this domain that blocks such activation also blocks apoptosis mediated by the Fas receptor (8). Thus, JNK may also play a role in apoptosis. For analysis of the JNK pathway, Promega offers the Anti-ACTIVE^® JNK pAb, Rabbit (Cat.# V7931 and V7932), which can be used in both Western analysis and immunocytochemistry.

References

1. Sun, X.M. et al. (1999) Effects of serum on calcium mobilization in the submandibular cell line A253. J. Biol. Chem. 274, 5053.
2. Juo, P. et al. (1998) Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade. Curr. Biol. 8, 1001.
3. Srinivasula, S.M. et al. (1996) Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases. Proc. Natl. Acad. Sci. USA 93, 14486.
4. Li, H. et al. (1998) Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94, 491.
5. Luo, X. et al. (1998) Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94, 481.
6. Li, P. et al. (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479.
7. Desagher, S. et al. (1999) Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis.  J. Cell Biol. 144, 891.
8. Chaudhary, P.M. et al. (1999) Activation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway by overexpression of caspase-8 and its homologs.  J. Biol. Chem. 274, 19211.
   

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