For the Greater Good
Apoptosis is the major form of programmed cell death in metazoan (multicellular) organisms. Programmed cell death is vital for proper development, tissue homeostasis, and immunity. This "altruistic" cell suicide is used as a response to various cellular stresses such as apoxia (extreme oxygen deprivation), irreparable genomic insults, viral infection, and to eliminate certain groups of cells such as those undergoing neoplastic transformations or self-targeting lymphocytes (agranular white blood cells which include natural-killer cells, T-cells, and B-cells). In order to successfully establish necessary pre-conditions for neoplastic transformations, cancers invest heavily in developing mechanisms to target and inactivate components of the apoptotic circuitry in order to evade elimination, and propagate uncontested. This section will introduce key components of the apoptotic circuitry and evaluate various "evasion mechanisms" utilized by numerous cancers.
A One-Family Wrecking Crew: The Caspases
Regardless of the death-inducing stimuli, apoptosis results in the fragmentation of several hundred proteins and DNA. This massive proteolysis is carried out by a family of cysteine aspartyl-specific proteases (or proteases specific for the c-terminal side of aspartic acid residues) termed caspases. Fragmentation of DNA is carried out by the caspase-induced DNase CAD (caspase-activated DNase) after caspases cleave the CAD chaperone/inhibitor ICAD (inhibitor of CAD). Free CAD digests chromatin into a characteristic ladder of nucleosomes.
Caspases (n = 11 in humans) are grouped according to structure and function. Initiator (or apical) pro-capsases (-8 or -9), have long pro-domains that target them to specific scaffold proteins following an apoptotic signal. Catalytically inactive pro-caspase -8 binds FADD/Mort1, while pro-caspase -9 binds the apoptosome which consists of mitochondiral cytochrome C bound to Apaf-1. Subsequent conformational changes activate the initiator caspases which then cleave the catalytically inactive pro-forms of the executioner (or effector) caspases (-3,-6,-7) to allow for assembly of catalytically active tetrameric proteases. Ultimately, the cascade of effector cascade activation results in cleavage of the inhibitor of caspase-activated DNase (ICAD) which liberates the DNase CAD to proceed with fragmentation of chromosomal DNA, ultimately killing the cell. Active caspases cleave ~1000 cellular substrates, some of which function to remove the cell "corpse" without invoking inflammation. This is in contrast to necrosis which does trigger apoptosis.
Many caspase-encoding genes are under transcriptional control of the E2F transcription factors, especially E2F1. E2F1 can increase transcription of several caspases 5-15 fold to highly sensitize the cell to pro-apoptotic stumuli, such as those emitted by p53. In addition, E2Fs also induce expression of genes encoding pro-apoptitic Bcl-2-related family members which sensitize the cell to apoptotic induction through intrinsic (mitochondial) apoptotic pathway discussed below.
A One-Family Wrecking Crew: The Caspases
Regardless of the death-inducing stimuli, apoptosis results in the fragmentation of several hundred proteins and DNA. This massive proteolysis is carried out by a family of cysteine aspartyl-specific proteases (or proteases specific for the c-terminal side of aspartic acid residues) termed caspases. Fragmentation of DNA is carried out by the caspase-induced DNase CAD (caspase-activated DNase) after caspases cleave the CAD chaperone/inhibitor ICAD (inhibitor of CAD). Free CAD digests chromatin into a characteristic ladder of nucleosomes.
Caspases (n = 11 in humans) are grouped according to structure and function. Initiator (or apical) pro-capsases (-8 or -9), have long pro-domains that target them to specific scaffold proteins following an apoptotic signal. Catalytically inactive pro-caspase -8 binds FADD/Mort1, while pro-caspase -9 binds the apoptosome which consists of mitochondiral cytochrome C bound to Apaf-1. Subsequent conformational changes activate the initiator caspases which then cleave the catalytically inactive pro-forms of the executioner (or effector) caspases (-3,-6,-7) to allow for assembly of catalytically active tetrameric proteases. Ultimately, the cascade of effector cascade activation results in cleavage of the inhibitor of caspase-activated DNase (ICAD) which liberates the DNase CAD to proceed with fragmentation of chromosomal DNA, ultimately killing the cell. Active caspases cleave ~1000 cellular substrates, some of which function to remove the cell "corpse" without invoking inflammation. This is in contrast to necrosis which does trigger apoptosis.
Many caspase-encoding genes are under transcriptional control of the E2F transcription factors, especially E2F1. E2F1 can increase transcription of several caspases 5-15 fold to highly sensitize the cell to pro-apoptotic stumuli, such as those emitted by p53. In addition, E2Fs also induce expression of genes encoding pro-apoptitic Bcl-2-related family members which sensitize the cell to apoptotic induction through intrinsic (mitochondial) apoptotic pathway discussed below.
Back Upstream: The Intrinsic (Mitochondrial) Apoptotic Pathway Triggers the Apoptotic Caspase-Cascade
The intrinsic (a.k.a. stress-activated) apoptotic pathway can be initiated within the cell by transcriptional activation (ex. via p53 or E2F1 transcription factors) of genes encoding pro-apoptotic Bcl-2-related family members (discussed below) which congregate at the outer surface of the mitochondrial membrane, causing release of cytochrome C. Numerous cell-physiologic stresses promote activation of specific pro-apoptotic Bcl-2 family members. Such stimuli include excessive intracellular calcium, excessive oxidizing agents (which is reduced as it removes electrons from a substrate which is then referred to as oxidized at the end of the red-ox reaction), DNA-damaging agents, agents that disrupt microtubule formation, and viral infection.
Mitochondrial cytochrome C is released from the space between in the inner and outer membranes of the mitochonrdia only after pro-apoptotic signals trigger depolarization (loss of normal voltage gradient) of the outer mitochondrial membrane. Cytochrome C spills out into the cytosol to associate with Apaf-1 (which is trans-activated by p53 or E2F1) and form a functional seven-spoked apoptosome which activates initiator pro-caspase-9 to trigger the apoptotic caspase cascade. The open mitochondrial membrane channel also releases other pro-apoptotic proteins such as Smac/DIABLO which antagonize the inhibitors of apoptosis (IAPs) that normally bind and inactivate the caspases.
Specialized channels on the mitochondrial outer membrane control the flow of Cytochrome C and several other proteins out into the cytosol. These membrane channels are regulated by members of the Bcl-2 (B-cell lymphoma-2 oncoprotein) family. "Pro-survival" Bcl-2 family members work to keep the mitochondrial outer membrane channels closed. These anti-apoptotic Bcl-2-related family members include "Bcl-2" family (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1). Each member of the Bcl-2 family has its own set of opposing family members. The "pro-death" Bcl-2-related family members attempt to open the mitochondrial outer membrane channels. These pro-apoptotic Bcl-2-related family members include the entire "Bax" family (Bax, Bak, and Bok) and the "BH3-only" family (Bid, Bim, Bik, Bad, Bmf, Hrk, Noxa, Puma). The human genome is known to encode 24 Bcl-2 related proteins, 6 of which are anti-apoptotic, while the remaining 18 are pro-apoptotic. Family members are grouped according to presence of certain functional domains, however every Bcl-2 related family member possesses a conserved amphipathic α-helical domain (BH3 domain) that binds a hydrophobic crevice on the surface of Bcl-2 and Bcl-XL.
Back Upstream: The Extrinsic (Receptor-Activated) Apoptotic Pathway Triggers the Apoptotic Caspase-Cascade
Homotrimeric ligands in the extracellular space bind and activate pro-apoptotic transmembrane cell surface receptors, often called death receptors. Subsequently, a cytoplasmic capsase cascade is activated which converges on the intrinsic apoptotic pathway. Trimeric death receptor ligands belong to the tumor necrosis factor (TNF) family of proteins, which includes TNF-α, TRAIL/APO2L, and Fas Ligand (FasL/CD95L). The human genome encodes 30 known members if the tumor necrosis factor (TNF)-family receptors which are grouped into 5 death receptor families: FAS, DR4, DR5, TNFR1, DR3. All TNF death receptors share a common "death domain" (DD) in their cytosolic tail.
Binding by the respective homotrimeric ligand causes death receptor trimerization, followed by binding and activation of FADD (FAS-associated death domain protein). FADD bound to the death domain of a death receptor is referred to as a DISC (death-inducing signaling complex). DISC recruits initiator pro-caspases 8 or 10, which both contain death effector domains (DED) in their N-terminal pro-domains that bind a corresponding DED in FADD.
DISC recruitment of inactive initiator pro-caspases 8 or 10 triggers self-cleavage of the pro-domain to form active initiator caspases which activate the executioner caspases 3, 6, and 7 to converge on the same apoptotic caspase cascade through which the intrinsic apoptotic program executes programmed cell death. The pro-apoptotic signal is amplified further when caspase 3 cleaves and inactivates the pro-apoptotic Bcl-2-related protein Bid which then migrates to the mitochondrial channel to release cytochrome C.
As previously mentioned, Cytochrome C spills out into the cytosol to associate with Apaf-1 (which is trans-activated by p53 or E2F1) and form a functional seven-spoked apoptosome. The oligomerized Apaf-1 of the apoptosome binds caspase-9 via its caspase recruitment domain (CARD), which activates initiator pro-caspase-9 to trigger the apoptotic caspase cascade. The open mitochondrial membrane channel also releases other pro-apoptotic proteins such as Smac/DIABLO which antagonize the inhibitors of apoptosis (IAPs) that normally bind and inactivate the caspases.
Cytotoxic T lymphocytes and natural killer (NK) cells trigger the extrinsic apoptotic program to kill target cells through activation of death receptors, or by secreting the enzyme granzyme B onto target cells. Granzyme B enters the target cell and cleaves and activates pro-capases 3, 8, and 9 to trigger the apoptotic caspase cascade.
Recommended Readings:
Mitochondrial Metabolic Pathways Cross-talk with Apoptotic Pathways (Review):
http://www.nature.com/onc/journal/v30/n38/full/onc2011167a.html?WT.ec_id=ONC-201109
Apoptosis-targeted therapies for cancer (Cancer Cell - Review 2003)
http://www.sciencedirect.com/science/article/pii/S1535610802002416
The intrinsic (a.k.a. stress-activated) apoptotic pathway can be initiated within the cell by transcriptional activation (ex. via p53 or E2F1 transcription factors) of genes encoding pro-apoptotic Bcl-2-related family members (discussed below) which congregate at the outer surface of the mitochondrial membrane, causing release of cytochrome C. Numerous cell-physiologic stresses promote activation of specific pro-apoptotic Bcl-2 family members. Such stimuli include excessive intracellular calcium, excessive oxidizing agents (which is reduced as it removes electrons from a substrate which is then referred to as oxidized at the end of the red-ox reaction), DNA-damaging agents, agents that disrupt microtubule formation, and viral infection.
Mitochondrial cytochrome C is released from the space between in the inner and outer membranes of the mitochonrdia only after pro-apoptotic signals trigger depolarization (loss of normal voltage gradient) of the outer mitochondrial membrane. Cytochrome C spills out into the cytosol to associate with Apaf-1 (which is trans-activated by p53 or E2F1) and form a functional seven-spoked apoptosome which activates initiator pro-caspase-9 to trigger the apoptotic caspase cascade. The open mitochondrial membrane channel also releases other pro-apoptotic proteins such as Smac/DIABLO which antagonize the inhibitors of apoptosis (IAPs) that normally bind and inactivate the caspases.
Specialized channels on the mitochondrial outer membrane control the flow of Cytochrome C and several other proteins out into the cytosol. These membrane channels are regulated by members of the Bcl-2 (B-cell lymphoma-2 oncoprotein) family. "Pro-survival" Bcl-2 family members work to keep the mitochondrial outer membrane channels closed. These anti-apoptotic Bcl-2-related family members include "Bcl-2" family (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1). Each member of the Bcl-2 family has its own set of opposing family members. The "pro-death" Bcl-2-related family members attempt to open the mitochondrial outer membrane channels. These pro-apoptotic Bcl-2-related family members include the entire "Bax" family (Bax, Bak, and Bok) and the "BH3-only" family (Bid, Bim, Bik, Bad, Bmf, Hrk, Noxa, Puma). The human genome is known to encode 24 Bcl-2 related proteins, 6 of which are anti-apoptotic, while the remaining 18 are pro-apoptotic. Family members are grouped according to presence of certain functional domains, however every Bcl-2 related family member possesses a conserved amphipathic α-helical domain (BH3 domain) that binds a hydrophobic crevice on the surface of Bcl-2 and Bcl-XL.
Back Upstream: The Extrinsic (Receptor-Activated) Apoptotic Pathway Triggers the Apoptotic Caspase-Cascade
Homotrimeric ligands in the extracellular space bind and activate pro-apoptotic transmembrane cell surface receptors, often called death receptors. Subsequently, a cytoplasmic capsase cascade is activated which converges on the intrinsic apoptotic pathway. Trimeric death receptor ligands belong to the tumor necrosis factor (TNF) family of proteins, which includes TNF-α, TRAIL/APO2L, and Fas Ligand (FasL/CD95L). The human genome encodes 30 known members if the tumor necrosis factor (TNF)-family receptors which are grouped into 5 death receptor families: FAS, DR4, DR5, TNFR1, DR3. All TNF death receptors share a common "death domain" (DD) in their cytosolic tail.
Binding by the respective homotrimeric ligand causes death receptor trimerization, followed by binding and activation of FADD (FAS-associated death domain protein). FADD bound to the death domain of a death receptor is referred to as a DISC (death-inducing signaling complex). DISC recruits initiator pro-caspases 8 or 10, which both contain death effector domains (DED) in their N-terminal pro-domains that bind a corresponding DED in FADD.
DISC recruitment of inactive initiator pro-caspases 8 or 10 triggers self-cleavage of the pro-domain to form active initiator caspases which activate the executioner caspases 3, 6, and 7 to converge on the same apoptotic caspase cascade through which the intrinsic apoptotic program executes programmed cell death. The pro-apoptotic signal is amplified further when caspase 3 cleaves and inactivates the pro-apoptotic Bcl-2-related protein Bid which then migrates to the mitochondrial channel to release cytochrome C.
As previously mentioned, Cytochrome C spills out into the cytosol to associate with Apaf-1 (which is trans-activated by p53 or E2F1) and form a functional seven-spoked apoptosome. The oligomerized Apaf-1 of the apoptosome binds caspase-9 via its caspase recruitment domain (CARD), which activates initiator pro-caspase-9 to trigger the apoptotic caspase cascade. The open mitochondrial membrane channel also releases other pro-apoptotic proteins such as Smac/DIABLO which antagonize the inhibitors of apoptosis (IAPs) that normally bind and inactivate the caspases.
Cytotoxic T lymphocytes and natural killer (NK) cells trigger the extrinsic apoptotic program to kill target cells through activation of death receptors, or by secreting the enzyme granzyme B onto target cells. Granzyme B enters the target cell and cleaves and activates pro-capases 3, 8, and 9 to trigger the apoptotic caspase cascade.
Recommended Readings:
Mitochondrial Metabolic Pathways Cross-talk with Apoptotic Pathways (Review):
http://www.nature.com/onc/journal/v30/n38/full/onc2011167a.html?WT.ec_id=ONC-201109
Apoptosis-targeted therapies for cancer (Cancer Cell - Review 2003)
http://www.sciencedirect.com/science/article/pii/S1535610802002416