What is apoptosis?


In the adult organism cell number is kept constant by carefully balancing cell division and cell death. Cells have to be replaced when they become old, diseased or infected but cell proliferation must by offset by cell death. If the equilibrium between cell proliferation and cell death is disturbed, for example if the cells divide faster than they die, tumor formation can occur, or conversely, if cells divide slower than they die this will result in an overall cell loss leading to hypotrophic or degenerative diseases.


Apoptosis is a form of programmed cell death in which the cell uses specialized cellular machinery to kill itself. This cell suicide mechanism enables metazoans to control cell number and eliminate cells that threaten the animal's survival. It involves a series of biochemical events that lead to a characteristic cell morphology which includes blebbing, loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation and eventual cell death. It is an important process in biology and its importance as a biological phenomenon has been demonstrated by its implication in an extensive variety of diseases.


Apoptosis often occurs when a cell is damaged beyond repair, infected with a virus, or is undergoing stressful conditions such as starvation. In these cases apoptosis functions to remove the damaged cell, preventing it from taking further nutrients from the organism, or halting further spread of viral infection. Apoptosis also plays a role in preventing cancer because if a cell is unable to undergo apoptosis because of mutation or biochemical inhibition, it continues to divide and can develop into a tumor.




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Apoptosis signalling pathways


 Apoptosis or programmed cell death is an evolutionarily conserved process that allows multicellular organisms to selectively discard superfluous or damaged cells through a highly orchestrated cell suicide programme. It is essential during embryogenesis and for maintaining cellular homeostasis. Apoptosis can be induced in response to cytokines, cytotoxic drugs, ionising radiation and reactive oxygen species (ROS) and is characterized by morphological changes including cytoplasmic shrinkage, membrane blebbing, nuclear condensation and fragmentation and finally fragmentation of the cell into membrane bound apoptotic bodies.


There are two distinct, yet interlinked, signalling pathways that control apoptosis activation. The intrinsic pathway is activated by intracellular cues, such as damage to the cell’s DNA while the extrinsic pathway is activated by extracellular signals, usually generated by cytotoxic cells of the immune system. Both pathways stimulate pro-apoptotic caspases. Caspases are synthesized as inactive pro-enzymes and are specialised, cysteine-dependent proteases that cleave cellular proteins, including cytoplasmic- and nuclear- cytoskeletal proteins, endonucleases and other caspases. In normal healthy cells caspases are present as inactive zymogens for protection, but can be activated by dimerization, autocleavage or processing by other proteases. Apoptotic signals first stimulate upstream initiator caspases (including caspases 8, 9 and 10) by recruiting them into specific signalling complexes that promote their multimerization. In turn, these activate downstream effector caspases (including caspases3, 6 and 7) by proteolytic processing. The effector caspases then process various cellular proteins, leading to the characteristic events of the apoptotic cell-death programme.


The Intrinsic Pathway


 The intrinsic pathway (fig.1) is initiated by internal stress such as growth factor deprivation, hypoxia, oxidative stress and DNA damage. It is controlled by the balance of pro- and anti-apoptotic Bcl2 family members. Pro –apoptotic members containing a single BH3 domain include Bid, Bad, Bim, Bmf, Puma and Noxa, while Bax, Bak and Bok are also pro-apoptotic but have multiple BH3 domains. In contrast Bcl-2, Bcl-X and Mcl-1 also have multiple BH3 domains but are anti-apoptotic. Interactions between the pro and anti-apoptotic BH3-multidomain proteins are modulated by the BH3-only proteins allowing a tight regulation of apoptosis induction. Mitochondria produce ATP by generating and maintaining an electrochemical gradient generated by the electron transfer chain thus driving ATP synthesis. This mitochondrial membrane potential is controlled by the mitochondrial permeability transition pore (mPTP) which transverses the two mitochondrial membranes and is under the control of the Bcl-2 protein family. Stress signals activate pro-apoptotic Bcl-2 family members. When activated, Bax and Bak form large oligomeric channels in the outer mitochondrial membrane leading to membrane permeabiliation and release of cytochrome  c. Once cytochrome c is in the cytosol, it binds to the WD40 domain of the apoptotic protease-activating factor Apaf-1 and to pro-caspase-9 resulting in the formation of the apoptosome complex, which in turn stimulates caspase9 and activates effector caspases. Other pro-apoptotic proteins such as endonuclease G (EndoG), second mitochondrial activator of caspases (SMAC/DIABLO) and apoptosis-inducing factor (AIF)) are also released in to the cytosol. AIF and EndoG translocate to the nucleus and induce chromatin fragmentation (Li et al., 2001). Omi/Htra2 and Smac/Diablo remain in the cytosol where they bind and inhibit the caspase inhibitory proteins (IAPs- inhibitor of apoptosis proteins) thereby promoting caspase activation.


 Extrinsic Pathway


Apoptotic cell death can be induced by the binding of death ligands to death receptors present on the surface of cells. Death ligands are primarily expressed on immune cells and function to aid the removal of damaged, infected or cancerous cells. The ligands belong to the greater cytokine tumour-necrosis factor (TNF) superfamily, and bind to pro-apoptotic ‘death’ receptors on the surface of the target cell, thereby activating the extrinsic apoptosis pathway. Ligation of death receptors by ligands initiates trimerisation of the death receptors and recruitment of adaptor molecules leading to formation of the death-inducing signalling complex (DISC) showm in Figure 1. Pro-caspase-8 (FLICE) molecules are then recruited leading to their dimerisation and autoactivation. Active caspase-8 activates downstream effector caspases like caspase-3 and -7 which commit the cell to death.


TRAIL-mediated apoptosis is an important strategy for anticancer therapy and has been studied extensively. TRAIL is a death ligand with a homotrimeric structure. Upon binding to its receptors DR4 or DR5, it induces receptor trimerization and a conformational change in the intracellular death domain (DD) resulting in receptor activation. Activation of the receptor allows the binding of the adaptor molecule, Fas-associated protein with death domain (FADD) via a homotypic, DD–DD interaction. FADD also contains a death effector domain (DED). The DED in FADD binds to the DED of pro-caspase-8/-10 resulting in their oligomerization and autoactivation .  Active caspase-8/-10 in turn activates the effector caspase that executes the apoptotic programme. Caspase-8 can also cleave and activate the pro-apoptotic Bcl-2 protein, Bid, which engages the intrinsic apoptotic pathway. Truncated Bid activates Bax and Bak, leading to their oligomerization and pore formation in the outer mitochondrial membrane. In some cell types the caspase-8/-10-triggered caspase cascade is sufficient to commit the cell to apoptosis, while in other cell types the intrinsic amplification loop is necessary for the commitment to apoptosis. Depending on the requirement for the intrinsic mitochondrial amplification loop for TRAIL-induced apoptosis, type I (independent) and type II (intrinsic mitochondrial amplification dependent) tumour cells can be distinguished.


 ER Stress


The endoplasmic reticulum (ER) comprises a complex membranous network found in all eukaryotic cells. It plays a crucial role in normal cellular functioning, particularly with regard to folding and post-translational modification of secretory proteins and membrane proteins, that are synthesized along the membrane of the rough ER and passed onto the Golgi apparatus for post-translational modifications, such as glycosylation and lipidation. In order to accomplish its protein folding functions the ER has high concentrations of chaperone proteins, which facilitate correct folding of nascent proteins. Many of these chaperones are Ca2+-dependent, and in fact the ER contains high concentrations of Ca2+ and plays an important role in intracellular Ca2+ homeostasis. The oxidizing environment that exists in the ER lumen is required for the formation of disulphide bonds during protein synthesis.


Disruption to ER function can occur as a result of a wide variety of cellular stress stimuli, including a number which are related to cancer and tumor development. These include expression of mutant proteins which cannot be correctly folded, depletion of energy supply to the ER (resulting from hypoxia and/or glucose deprivation), changes to the redox environment (e.g., resulting from hypoxia), viral infection and Ca2+ depletion. All of these cause disruption of protein folding capacity and lead to the build-up of unfolded and misfolded proteins within the ER. The cell responds by shutting down global protein synthesis, and initiating mechanisms to deal with the backlog of proteins, i.e., enhanced protein folding capacity through selective expression of ER chaperone proteins, and enhanced degradation of misfolded proteins within the ER through a process called ER-associated degradation (ERAD). This co-ordinated biochemical response to the accumulation of unfolded and/or misfolded proteins within the ER is termed the unfolded protein response (UPR).


The unfolded protein response is mediated through the activation of three ER transmembrane stress sensors (Fig. 1). These are pancreatic ER kinase (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1). These proteins are maintained in an inactive state through a physical interaction between their ER lumen domains and the ER chaperone glucose-regulated protein of 78 kDa (GRP78). An increase in the level of unfolded proteins within the ER triggers redirection of GRP78 to these unfolded proteins, which consequently releases and activates the ER stress sensors, and launches the unfolded protein response.


The unfolded protein response and survival


Initially activation of the three arms of the unfolded protein response promotes pro-survival signaling. The unfolded protein response causes the induction of chaperone proteins that increase the protein folding capacity of the ER. Upon dissociation from GRP78, PERK homodimerizes and becomes autophosphorylated, generating active PERK. One of the main targets of active PERK is eukaryotic initiation factor 2α (eIF2α), whose phosphorylation leads to inhibition of cap-dependent protein translation. This rapidly reduces the load of nascent proteins being channelled into the ER. Cap-independent translation persists however, allowing the synthesis of key proteins that mediate the unfolded protein response itself. One such protein is ATF4, a transcription factor which induces ER chaperones (e.g., GRP78 and GRP94), proteins involved in regulation of amino acid metabolism and resistance to oxidative stress(Harding et al., 2003) which promote survival, in addition to the transcription factor C/EBP homologous protein (CHOP) which is known to promote apoptotic cell death. Thus, PERK activation is initially protective, although it later promotes cell death. Dissociation of the second ER stress sensor ATF6 from GRP78 triggers its translocation to the Golgi apparatus, where it is cleaved to its active form by specific proteases (SP-1 and SP-2). Active ATF6 can then translocate to the nucleus, where it induces transcription of genes including GRP78, GRP94, protein disulphide isomerase (PDI) and X box-binding protein 1 (XBP1). These proteins function to counteract ER stress and act in a pro-survival capacity. The third ER stress sensor, IRE1, acts both as a serine-threonine protein kinase and an endoribonuclease. Once active, IRE1 is responsible for the unconventional splicing of XBP1 mRNA, whose transcription is up regulated by the ATF6 arm of the unfolded protein response. This alternatively spliced XBP1 encodes a transcription factor that targets diverse genes including ER chaperones and P58IPK, the latter of which binds and inhibits PERK, providing a negative feedback loop. If activation of the unfolded protein response is successful in removing the accumulation of misfolded proteins in the ER, then normal protein translation is resumed and the cell recovers.

The ER lumen houses a large array of proteins involved in different aspects of protein folding and degradation, including GRP78/BiP, GRP170/ORP150, GRP58/ERp57, PDI, ERp72, calnexin, calreticulin, ER degradation-enhancing alpha-mannosidase-like protein (EDEM), Herp and co-chaperones SIL1 and P58IPK. Of these, the chaperone protein GRP78 is the best characterized. It is a member of the highly conserved Hsp70 protein family, which is constitutively expressed, and is up regulated by unfolded protein response activation. As previously mentioned, increased expression of GRP78 enhances the protein folding capacity of the ER, and is associated with pro-survival responses. In fact, reduction of GRP78 expression with siRNA activates the unfolded protein response and leads to apoptosis in HeLa cells. Conversely, since GRP78 normally binds to the three ER stress sensors, it is likely that its increased expression could promote enhanced survival of cells, and would block unfolded protein response activation. One protective function of GRP78 could relate to its interaction with components of pro-apoptotic pathways emanating from ER. For example, GRP78 could inhibit apoptosis by binding and blocking caspase-7 and caspase-12 activation, or pro-apototic proteins Bik and Bax and prevent cytochrome c release from mitochondria. An inability to activate the unfolded protein response would initially mean lack of PERK, ATF6 and IRE1 activation, and of their downstream pro-survival effectors. However, it would also mean that the cell could not activate pro-apoptotic signaling even when there is severe ER stress(discussed below).


The unfolded protein response and cell death


Although the unfolded protein response is primarily a pro-survival response, in the event of prolonged or severe ER stress that is not resolved, the unfolded protein response switches to initiation of apoptosis. The biochemical mechanisms responsible for the switch are not yet clear, and are reviewed in Szegezdi et al., 2006 EMBO Rep.Sep;7(9):880-5). Certain components of the unfolded protein response are strongly linked to induction of ER stress-induced apoptosis. In this regard, CHOP, which is induced by all three arms of unfolded protein response through XBP1, ATF4 and ATF6 transcription factors, is required for ER stress-induced apoptosis in many instances. CHOP alters the balance between pro-survival and pro-apoptotic Bcl-2 family members thus promoting apoptosis through the mitochondrial pathway. For example, CHOP causes upregulation of Bim, a pro-apoptotic BH3-only member of the Bcl-2 family  and suppresses anti-apoptotic proteins. It has also been reported that CHOP causes upregulation of TRAIL receptors and thus promotes the extrinsic apoptotic pathway in certain cancer cells. Apart from CHOP, other mechanisms of apoptosis initiation by ER stress have been described. Active IRE1 recruits the adaptor molecule TNF receptor-associated factor 2 (TRAF2), which further recruits apoptosis-signal-regulating kinase (ASK1) leading to activation of c-jun N-terminal kinase (JNK). JNK signaling is known to influence the cell death machinery through the phosphorylation-mediated regulation of Bcl-2 family members. IRE1-TRAF2 has also been reported to recruit pro-caspase-12, an ER-associated caspase that has been proposed as a key mediator of ER stress-induced apoptosis. However, while caspase-12 is expressed in rodents, a human orthologue has not been identified to date, and substrates for this caspase have not been identified. The cytosolic domain of IRE1 has also been shown to form a complex with proapoptotic proteins which are essential for IRE1 activation. Thus, proapoptotic proteins function at the ER membrane to activate IRE1 signaling and to provide a physical link between members of the core apoptotic pathway and the unfolded protein response.

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