1. Programmed Cell Death Programmed Cell Death
Programmed cell death (PCD), or apoptosis, can be triggered by a wide range of stimuli, including cell surface receptors like Fas or tumor necrosis factor receptor 1 (TNFR1). It constitutes a system for the removal of unnecessary, aged, or damaged cells that are regulated by the interplay of proapoptotic and antiapoptotic proteins of the Bcl-2 family. The proapoptotic proteins Bax, Bad, Bid, Bik, and Bim contain an a-helical BH3 death domain that fits the hydrophobic BH3 binding pocket on the antiapoptotic proteins Bcl-2 and Bcl-XL, forming heterodimers that block the survival-promoting activity of Bcl-2 and Bcl-XL. Thus, the relative abundance of proapoptotic and antiapoptotic proteins determines the susceptibility of the cell to programmed death. The proapoptotic proteins act at the surface of the mitochondrial membrane to decrease the mitochondrial transmembrane potential and promote leakage of cytochrome c. In the presence of dATP, cytochrome c complexes with and activates Apaf-1. Activated Apaf-1 binds to downstream caspases, such as pro-caspase-9, and processes them into proteolytically active forms. This begins a caspase cascade resulting in apoptosis. Smac/Diablo is released from the mitochondria and blocks IAP proteins which normally interact with caspase-9 to inhibit apoptosis. The lower panel shows the conserved apoptotic pathway in C. elegans. CED-3 encodes a caspase whose function is facilitated by CED-4, which is highly similar to Apaf-1. CED-4 function is blocked by CED-9, which protects cells against apoptosis and is similar to the human antiapoptotic protein Bcl-2. CED-9 activity is inhibited by EGL-1, which is similar to the proapoptotic Bcl-2 family members.

2. Mitochondria in Apoptosis
Oxidative damage due to the endogenous production of reactive oxygen species by mitochondria can lead to cell death via apoptosis or necrosis. Increases in cytosolic Ca2+ levels due to ion channel-linked receptors, such as the excitatory amino acid neurotransmitter glutamic acid, can induce permeability transition (PT) in the mitochondrial membrane. PT constitutes the first rate-limiting event of the common pathway of apoptosis. Upon PT, apoptogenic factors leak into the cytoplasm from the mitochondrial intermembrane space. Two such factors, cytochrome c and apoptosis inducing factor (AIF), begin a cascade of proteolytic activity that ultimately leads to nuclear damage (DNA fragmentation, DNA mutations) and cell death. Cytochrome c, a key protein in electron transport, appears to act by forming multimeric complexes with Apaf-1, a protease, which in turn activates pro-caspase-9, and begins a cascade of activation of downstream caspases. Bcl-2 and Bcl-X can prevent pore formation and block the release of cytochrome c from the mitochondria and prevent activation of the caspase cascade and apoptosis. PT is also related to the mitochondrial generation of reactive oxygen species, which play a role in the degradation phase of apoptosis (i.e. plasma membrane alterations). In contrast, high levels of oxidative stress can lead to PARP activation. Activated PARP causes conformational changes in the chromatin structure allowing the recruitment of DNA repair proteins or other transcription factors. This process consumes large amounts of NAD+, effectively causing a depletion of ATP leading to necrosis.

3. Caspase Cascade Caspase Cascade
Apoptosis or programmed cell death is triggered by a variety of stimuli, including cell surface receptors like FAS, the mitochondrial response to stress, and factors released from cytotoxic T cells. The caspases comprise a class of cysteine proteases, many members of which are involved in apoptosis. The caspases convey the apoptotic signal in a proteolytic cascade, with caspases cleaving and activating other caspases that in turn activate enzymes that subsequently degrade cellular targets that lead to cell death. The activating caspases include caspase-8 and caspase-9. Caspase-8 is the initial caspase activated in response to receptors with a death domain that interacts with FADD. The mitochondrial stress pathway begins with the release of cytochrome c from mitochondria, which then interacts with Apaf-1, causing self-cleavage and activation of caspase-9. The effector caspases, caspase-3, -6, -7 are downstream of the activator caspases and act to cleave various targets. Activation of caspase-3 and caspase-7 can be blocked by IAP proteins. Granzyme B and perforin, proteins released by cytotoxic T cells, induce apoptosis in target cells by forming transmembrane pores and triggering apoptosis, perhaps through cleavage of caspases. Caspase-independent mechanisms of granzyme B-mediated apoptosis have been suggested. In addition, caspase-activated DNAse (CAD) may be activated through the cleavage of its associated inhibitor ICAD. CAD is then able to interact with components such as topoisomerase II (Topo II) to condense chromatin and lead to DNA fragmentation.

4. Granzyme B
Granzyme B
Granzyme B and perforin, proteins released by an effector cell (cytotoxic T cell), can induce apoptosis in target cells by forming transmembrane pores and through cleavage of effector caspases such as caspase-3. In addition, caspase-independent mechanisms of granzyme B-mediated apoptosis have been suggested. Caspase-activated DNAse (CAD) is activated through the cleavage of its associated inhibitor ICAD by caspase-3. CAD is then able to interact with components such as topoisomerase II (Topo II) to condense chromatin, leading to DNA fragmentation and ultimately apoptosis.

5. Fas Signaling Pathway Fas Signaling Pathway
Fas/APO-1/CD95 (36 kDa) is a member of the tumor necrosis factor (TNF) receptor superfamily, a family of transmembrane receptors that also includes p75 neurotrophin receptor, TNFRI, and a variety of other cell surface receptors. Fas has been shown to be an important mediator of apoptotic cell death, as well as being involved in inflammation. Binding of the Fas ligand (Fas-L) induces trimerization of Fas in the target cell membrane. Activation of Fas causes the recruitment of Fas-associated protein with death domain (FADD) via interactions between the death domains of Fas and FADD. Pro-caspase-8 binds to Fas-bound FADD via interactions between the death effector domains (DED) of FADD and pro-caspase-8 leading to the activation of caspase-8. Activated caspase-8 cleaves (activates) nine other pro-caspases, in effect beginning a caspase cascade that ultimately leads to apoptosis. Caspases cleave nuclear lamins, causing the nucleus to break down and lose its normal structure. Fas-induced apoptosis can be effectively blocked at several stages by either FLICE-inhibitory protein (FLIP), by Bcl-2, or by the cytokine response modifier A (CrmA). Moreover, caspase-8 can activate Bid which is then able to associate with the mitochondria and promote leakage of cytochrome c. In the presence of dATP, cytochrome c complexes with and activates Apaf-1. Activated Apaf-1 binds to downstream caspases, such as pro-caspase-9, and processes them into proteolytically active forms. This begins a caspase cascade resulting in apoptosis. In addition, Smac/Diablo is released from the mitochondria and blocks IAP proteins which normally interact with caspase-9 to inhibit apoptosis.

6. TNF Signaling Pathway TNF Signaling Pathway
When bound to tumor necrosis factor (TNF), the TNF receptor (TNFR) (55 kDa) transduces growth regulatory signals into the cell. TNF is mitogenic for normal cells; however, TNF initiates programmed cell death (PCD) or apoptosis in transformed cells causing DNA fragmentation and cytolysis. Functional studies have identified a conserved region within the receptor, termed the death domain (DD), a protein-protein interaction motif that is necessary to transmit the apoptotic signal. Upon ligand binding, TNFR trimerizes and recruits the adaptor protein TRADD, as well as other death domain containing proteins such as RIP, RAIDD, and FADD. The recruitment of these proteins allows for activation of a caspase cascade and apoptosis. On the flip side, the TNF-induced cell survival pathway is mediated by the transcription factor NF-kB. Activation of NF-kB occurs via phosphorylation of IkB at Ser32 and Ser36, resulting in the dissociation and subsequent nuclear localization of active NF-kB. Recent studies have demonstrated that cells in which the NF-kB signaling pathway is blocked are more likely to undergo apoptosis in response to TNF. Therefore, the availability of NF-kB may play a critical role in the ability of TNF to act as an apoptosis-inducer and anti-tumor agent.

7. ATM/p53 Signaling Pathway
The ataxia telangiectasia-mutated gene (ATM) encodes a protein kinase that acts as a tumor suppressor. ATM activation, via IR damage to DNA, stimulates DNA repair and blocks cell cycle progression. One mechanism through which this occurs is ATM-dependent phosphorylation of p53. p53 can cause growth arrest of the cell at a checkpoint to allow for DNA damage repair or can cause the cell to undergo apoptosis if the damage cannot be repaired. The critical role of p53 is evident by the fact that it is mutated in over 50% of all human cancers.

8. Integrin Signaling in Cell Survival and Death
Integrins are heterodimeric transmembrane receptors composed of a- and b-subunits. Approximately 20 integrins have been identified thus far with a subset of them shown to support adhesion-dependent growth factor activation of the MAPK pathway. Upon ligand binding, integrin signaling leads to the tyrosine phosphorylation of cytoskeletal and other signaling components. Focal adhesion kinase (FAK) is activated via autophosphorylation when cells interact through integrins. A large array of other signaling molecules that promote cell survival bind to FAK and are phosphorylated, including Grb2/Sos and p85 of the PI3 Kinase. Thus, depending on the integrin interactions, the cell can either survive or undergo apoptosis.

9. Activation and Inhibition of Apoptosis
Several mechanisms have been identified in mammalian cells for the induction of apoptosis. These mechanisms include factors that lead to perturbation of the mitochondria leading to leakage of cytochrome c or factors that directly activate members of the death receptor family. Fas is a member of the tumor necrosis factor (TNF) receptor superfamily, a family of transmembrane receptors that include neurotrophin receptor (p75NTR), TNF-R1, and a variety of other cell surface receptors. Fas Ligand (Fas L) transmits signals to Fas on a target cell by inducing trimerization of Fas. Activation of Fas causes the recruitment of Fas-associated protein with death domain (FADD) via interactions between the death domain of Fas and FADD and is followed by pro-caspase-8 binding to FADD via interactions between the death effector domains (DED) of FADD and pro-caspase-8 leading to the activation of caspase-8. Activation of caspase-8 leads to the activation of other caspases, in effect beginning a caspase cascade that ultimately leads to apoptosis. Caspase-8 activation can also activate Bid, leading to activation of the apoptotic program. Fas-induced apoptosis can be effectively blocked at several stages by either FLICE-inhibitory protein (FLIP), by Bcl-2, or by the cytokine response modifier A (CrmA). In addition, activation of caspase-3 by caspase-9 can be blocked by inhibitor of apoptosis proteins (IAPs). Moreover, the protein kinase, Akt, can be activated by various growth factors and its activity can be blocked by PTEN. Akt functions to promote cell survival through two distinct pathways. Akt inhibits apoptosis by phosphorylating the Bcl-2 family member Bad, which then interacts with and dissociates from Bcl-XL allowing for cell survival. Alternatively, Akt activates IKK- that ultimately leads to NF-B activation and cell survival. Proapoptotic Bcl-2 family members, such as Bax and Bak can promote mitochondrial permeability, while Bcl-2 can inhibit their effects. Upon mitochondrial permeability, apoptogenic factors are released from the mitochondrial inter-membrane space and leak into the cytosol. One factor is cytochrome c, which induces the liberation of protease activators (caspases) that ultimately lead to apoptosis through nuclear damage (DNA fragmentation, DNA mutations). In addition, Smac/Diablo is released and can block IAP inhibition of capsase activity. Mitochondrial permeability is also related to the increased generation of reactive oxygen species (ROS), which plays a role in the degradation phase of apoptosis (i.e. plasma membrane alterations).

10. Caspase Activation Intristic Pathway
Caspase Activation Intristic Pathway Cytochrome c is released from the mitochondria of pre-apoptotic cells and binds to Apaf-1 in the presence of dATP/ATP. The interaction results in a conformational change in Apaf-1 allowing the molecules of Apaf-1 to associate with each other. This formation results in a wheel-like structure that contains 7 molecules each of Apaf-1, cytochrome c and ATP. Pro-Caspase-9 contains a caspase recruitment domain (CARD) that is used to mediate specific interactions with Apaf-1 CARD, which becomes exposed on the apoptosome during assembly. Pro-Caspase-9 is autoactivated and after cleavage, mature caspase-9 remains bound to the apoptosome where it is able to activate executioner caspases such as caspase-3 and caspase-7.