Molecular Mechanisms of Apoptosis

We are just beginning to unravel the complexities and intricacies of the regulation of apoptosis. Insight has developed rapidly in the last decade from (1) studies on cytokine- and chemotherapeutic agent-induced cell death, (2) genetic regulation of cell death in the nematode C. elegans,11 and

(3) studies on proapoptotic tumor suppressor genes such as p53 and antiap-optotic oncongenes, most notably bcl-2.12

Control of apoptosis is possible at many levels. This regulation can be expressed as a positive or negative modulating effect (Table 1.1): transcriptional regulation, induction of early intermediate genes; stage of the cell cycle


Positive and Negative Modulators of Apoptosis


Positive and Negative Modulators of Apoptosis









Baculovirus p35

Bag, Bak, Mcl-1, Bok

Cowpox virus serpin crm A

TNF superfamily (Fas, TNFR-1, Reaper)


Chemotherapeutic agents







IL-6, IL-3, erythropoetin


and relative levels of cyclins; presence or absence of nerve growth factor and its receptors; TNF-a and related receptors13; Fas-Fas-L interactions,14 ceram-ide as proapoptotic lipid second messenger and the sphingomyelin cycle15; the neuroprotective bcl-2 oncogene and its homologues,16 p53 and retinoblastoma genes as inducers of growth arrest and apoptosis17; the early initiator and later executionary caspase cascades and their triggers and inhibitors18; the role of the mitochondrion as central processor of incoming messages, and the role of translocation of inner mitochondrial membrane proteins such as cytochrome c, Apaf-1, and other factors to be found.19 A hypothetical and simplified choreography depicting possible interactions, as best illustrated with apoptosis-inducing cytokines, is outlined in the scheme shown. According to this model, the action of proapoptotic cytokines, such as TNF, Fas-L, or NGF, on their membrane receptors (P75 receptor in the case of NGF) results in recruitment/activation of a number of adapter proteins such as FADD, TRAFs, and TRADs. These proteins, though poorly understood mechanisms, couple the occupied receptors to distinct pathways of signaling and cell regulation. Whereas Fas appears to be a more dedicated proapop-totic receptor, the TNF receptors couple to apoptotic, antiapoptotic, and inflammatory pathways. Thus, TNF can activate the following: (1) NF-kB, which predominantly functions as antiapoptotic transcription factor; (2) the jun kinase (JNK) or stress-activated kinase (SAPK) pathway, which primarily functions in the regulation of stress, at times promoting apoptosis and at other times inhibiting it; and (3) the MACH/Flice protease, a member of the caspase family of proteases, which launches the apoptotic functions of TNF.20

It is not yet clear how MACH/Flice turns on the apoptotic program. In the case of Fas, it has been proposed that a cascade of proteases is turned on, and that it is necessary and sufficient to cause apoptosis. This proposed mechanism now appears as an over-simplified explanation, especially in the case of TNF, where many endogenous pathways are activated and regulated in response to TNF and Fas and contribute to the terminal apoptotic outcome. These pathways include the formation of reactive oxygen intermediates and changes in mitochondrial permeability and function.21 Also implicated are ceramide- and sphingolipid-derived molecules as stress-induced mediators that promote and enhance the apoptotic program.

Noncytokine stresses, such as heat, oxidative damage, and DNA-damag-ing agents also activate apoptosis by generating poorly understood internal signals. It is not yet determined whether these processes overlap cytokine-induced apoptosis, but in the case of DNA-damaging agents the proapop-totic protein P53 plays an important role in driving the response of the cells either through induction of cell cycle arrest or the induction of apoptosis.22

Significant results now implicate cytochrome c as a key mediator of the apoptotic pathways (Figure 1.1). Many, but not all, inducers of apoptosis cause the release of cytochrome c from the mitochondria. Also, it is now assumed that the mitochondrial membrane is the site of action of members of the Bcl-2 family of pro- and antiapoptotic proteins.22 It is suggested that bcl-2, the mammalian homologue of the ced-9 gene from C. elegans, functions primarily by inhibiting the release of cytochrome c, whereas proapoptotic relatives of bcl-2 may promote this event. The released cytochrome c interacts with Apaf-1, a positive regulator of apoptosis with homology to the C. elegans ced-4 proapoptotic gene. This collaboration results in activation of downstream caspases such as caspase 3, which are homologues of the C. elegans ced-3 gene. It is the action of these caspases on their substrates that results in the systematic degradation of key substrates such as nuclear lamins, PARP, fodrin, protein kinases, and other structural or regulatory proteins. This process culminates in the organized collapse of the nucleus, membranes, and cellular organelles. Many neuronal proteins are now recognized as substrates of caspases, including presenilins and huntingtin.23,24 The orderly breakdown of dying cells through the apoptotic mechanisms results in the packaging of cellular debris into apoptotic bodies which are then cleared by reticuloen-dothelial cells as well as normal adjacent cells, thus preventing inflammatory reactions to cell fragments.

The study of existing apoptotic developmental and neurodegenerative diseases, be they caused by a genetic defect or a triggering environmental factor, provide us with naturally occurring human models that validate existing hypotheses in neuronal culture systems and provide new information pertinent to basic cell biology.

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