Apoptosis could be activated by two major mechanisms (Kuo, Y. C. at al 2006); the intrinsic pathway which involves mitochondria related apoptosis and the extrinsic pathway which involves the death inducing signaling complex (DISC) (Green and Reed, 1998; Sheikh and Huang, 2004). Many apoptotic stimuli that induce metabolic stress in cell organelles include fragmentation of the endoplasmic reticulum and remodeling the cristaceae as early positive signals for intrinsic apoptosis. But must of all apoptotic signaling will eventually converge on the mitochondria apoptotic death pathway (Philchenkov A, 2004) leading finally the cell to die due to its dysfunction. Not all apoptotic events necessary involves the activation of the caspases for its execution but it does compromises the permeabilization of the mitochondrial membrane as the pivotal biochemical indicator and initial hallmark of activation (Constantini P et al, 2000; Green, D. R. and Reed, J. C., 1998; Skulachev, 1996).

On the intrinsic apoptotic pathway, mitochondria have been pointed out to be centrally responsible as an early indicator of apoptosis. Various inducers of apoptosis can directly or indirectly influence disruption of the organelle affecting the permeability of the outer mitochondrial membrane ultimately leading to the completion of apoptosis. Previous to cell lysis, permeabilization of both mitochondrial membranes and the loss of inter-transmembrane potential (ITP) is a first response for disruption in regular and cancerous cells (Constantini P et al, 2000). The dissipation in the ITP (ΔΨm) causes the permeabilization of mitochondrial membranes (Constantini P, 2000; Xiang J, 1996; Pastorno J G, 1998; Hirsch T, 1997; Brunet C L, 1998; Berndt C, 1998, Zamazami N., 1996; Vander Heiden M. G., 1997; Bossy-Wetzel G., 1998) pursued by the liberation of apoptogenic proteins such as cytocrome c (Shinohara Y. et al 2002), apoptosis induction factor (AIF) (Constantini P., 2000; Li J. D. et al, 2003) and smac/DIABLO (Karbowski M. et al, 2003). The liberation provokes a domino effect leading the apoptosis cascade by the activation of other pro-apoptotic proteins and inactivation of anti-apoptotic proteins (Letai A. et al, 2002; Karbowski M. et al, 2003).

Death receptors localized on the exterior of the cell (such as Fas, TNF and TNFR1) act as upstream activators of the extrinsic pathway. Opposed to TNF, Fas ligand receptor is thought to be the simplest, direct and most specific apoptotic extrinsical upstream activator that subsequently desencadenantes the caspases cascade ( Timmer et al, 2002).

Caspases are a family of proteins from the cysteine proteases that become activated upon apoptosis, they break down cellular components that cause fragmentation of nuclear DNA into nucleosomal units upon activation of caspase activated Dnase (CAD). The apoptotic caspases cascade is a network of interconnected initiators and effectors that order the main event of the execution. The most important and revised caspases are the initiator caspases which are 8, 9 and 10 and the effectors that are 3 and 7. Caspases 8 and 10 become activate through the Death Inducing Signaling Complex (DISC) (Fas ligand). Caspase 9 becomes activated through the permeabilization of the mitochondrial membrane which releases cytochrome c, Apaf-1 and pro-caspase 9. (Scorrano L, 2005). Subsequently caspase 9 could activate caspase 3 and 7 as a downstream event Caspases 3 and 7 become activated through DISC, the permeabilization of the mitochondrial membrane.

Now, caspases 8 and 10 could converge with the intrinsic mitochondrial pathway activating also apoptosis parallel to the extrinsic and downstream activating caspases 3 and 7 Both intrinsic and extrinsic pathways include activation of the mitochondria apoptotic-dependent (intrinsic) death pathway by permeabilization of the inter-membranes leading to cytoplasmic efflux of cytochrome c as key events (Philchenkov A, 2004).

Hello, I have been working with both extrinsic and intrinsic apoptotic pathway. To induce the extrinsic pathway I have used Fas mAb (really effective), for the intrinsic staurosporine. At one point the pathways overlap (i.e caspase 3 is activated in both cases) but for the extrinsic pathway you can measure caspase 8, for the intrinsic caspase 9 with specific fluorogenic substrates. For more info about concentrations to use and kinetic check my paper:

AIF and Scythe (Bat3) Regulate Phosphatidylserine Exposure and Macrophage Clearance of Cells Undergoing Fas (APO-1)-Mediated Apoptosis

Cheers,

Giulio

Hello, to check intrinsic pathway you can measure Bcl2 proteins, smac omi, AIF, APAF1 etc.  and for extrinsic you can check caspase 8 and death receptors such as TNFR etc.