Apoptosis assay

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Apoptosis


Suicide and homicide: apoptosis and necrosis

If programmed cell death can be taken as suicide in which cells kill themselves in certain physiological or pathological conditions, necrosis can be taken as homicide. The killer is high temperature, low oxygen, nutrition deficiency, or other physical or chemical factors. As for inner cells, the suicide and the homicide are obviously different especially in morphology (see below).

In apoptosis cytoplasm, nucleus and membranes change with variation of biochemical and physical factors. In early stage of apoptosis, cells expand and turn round, detach with adjacent cells and shrink. In cytoplasm, endoplasmic reticulum expands and turns to vacuole. In nucleus, chromatin condensates crescently around the nuclear membranes and becomes more basophilic. Eventually nucleus cracks into fragments that are wrapped by nuclear membranes. On cellular membranes, cells detach and membranes become more active and invaginate. These changes lead to the conversion from intact cell to apoptotic bodies in which contains fragmented cellular ingredients. Under physiological conditions, certain regulatory reactions occur on cell membranes which induce apoptotic bodies recognized and digested by phagocytes without raising inflammatory responses.

Autophagy is a process in which some of cytoplasm and organelles undergo series of degradations in lysosomes. It is featured with that phagosomes are formed in small vacuoles, ER disperses and chromosome moderately condensates. Although cytoskeleton disaggregates in autophagy, the cytoskeletal degradation is less obvious than that happened in apoptosis.

Necrosis leads to expansion of organelles like mitochondria, breaks of membranes and severe loss of cellular ingredients with inflammatory responses in surrounding tissues. In some cases, it’s easy to distinguish apoptosis and necrosis. However, in some cases it’s almost impossible to make sure whether cells are killing themselves or being killed, because dying cells have characteristics of both of apoptosis and necrosis.

Mechanism: signal transduction pathways

Like any other molecular biological mechanisms, mechanism of apoptosis is complicated and involves many molecules. As mostly accepted there are two styles of apoptosis: death receptors induced apoptosis or extracellular factors induced apoptosis, and mitochondria induced apoptosis or intracellular factors induced apoptosis. Though the upstream events of the two apoptosis styles are different, they both lead to activation of caspases. In normal cells apoptosis is strictly controlled and the caspases are in an unactivated pro-enzyme state. When caspases are activated which represents the apoptosis begins, it occurs the cascade reactions and triggers the irreversible apoptosis.

  • Extracellular factors-induced apoptosis:

It refers to that certain death ligands (e.g. Fas ligands, TNF-α and TRAIL) interact with the death receptors on the cell surface (e.g. members of TNF [tumor necrosis factor] super family) to induce apoptosis. Different signaling pathways activated by different death receptors are similar.Binding of receptors and ligands leads to generation of ceramide. The release of ceramide is then considered to accelerate the fusion of lipid rafts and gathers the death receptors. This aggregation is thought to amplify the signal of the apoptosis. Though in some cells such as lymphocytes, aggregation of receptors doesn’t occur, it triggers apoptosis. In most cases, amplification of the signal transduction pathways is necessary to activate an typical apoptosis process.

After binding with ligands a receptor makes a conformational change and the “death domain” exposes. At the same time different apoptosis proteins are recruited. This protein complex is called DISC (death-inducing signaling complex). At last it recruits a specific kind of caspases, normally precursor of caspase-8, and leads to activation of caspase-8 and initiation of apoptosis.

Binding of TNF-α to TNF Receptor-1 leads to tri-polymerization and intracellular aggregation of death domains. After the conformational change, TNF R-1 recruits an adaptor protein called TRADD (TNFR- associated death domain). TRADD then recruits more different proteins such as downstream TRAF 2 (TNF-associated factor 2), and activates NF-κB and JNK signaling pathways. TRADD can bind to FADD (Fas-associated death domain protein) and recruit precursor of caspase-8 via protein-protein interactions and forms the DISC complex. In process of DISC formation, precursors of caspase-8 break into active caspase-8 which then is released to cytoplasm and triggers downstream cascade reactions. FADD apoptosis pathway can be inhibited by FLIP(Flice-inhibitory protein). FLIP is analogous to caspase-8 and lack of proteolytic activity. It can depress the apoptosis by inhibiting the interaction of FADD and precursor of caspase-8.

The pathway by which ligands of Fas (FasL or CD95) activate apoptosis is similar to that of TNF. The binding of ligands accelerates aggregation of receptors, formation of DISC and activation of caspase cascade reactions. However, signaling pathway of Fas receptor is simpler. Adaptor protein FADD directly binds to death domain of Fas receptor, without presence of TRADD. Fas receptors seem to function only in apoptosis, unlike that TNF receptors also participate in other signaling pathways. As the same, this process can be inhibited by FLIP.

In cells that response to Fas, type Ⅰ cell such as thymocyte, caspase-8 is sufficiently activated by Fas and leads to apoptosis. In these cells high level Bcl-2 can’t inhibit apoptosis that induced by Fas. In type Ⅱ cells such as hepatocyte, caspase-8 is not sufficiently activated by Fas, thus apoptosis signals in these cells need to be amplified via apoptosis mitochondria. Activated caspase-8 leads a cleavage of cyplasmic Bid and turns it to tBid (truncated Bid). tBid enters into mitochondria and releases cytochrome C which then amplifies apoptosis signals.

In many cells, TRAIL (TNF-related apoptosis inducing ligand) can bind to receptor DR4 and DR5 and triggers rapid apoptosis. Intriguingly, there are some decoy receptors that compete with DR4 and DR5. These decoy receptors are called DcR1 and DcR2. Both of them can bind to TRAIL ligands and do not trigger apoptosis, because DcR1 doesn’t have intracellular domain and the death domain of DcR2 is truncated for that four of the six amino acids which are necessary for recruiting the adaptor proteins are missed.

NF-κB is an inhibitor of apoptosis. Typically, NF-κB binds inhibitor IkB which presents in cytoplasm. With inducement of TNF, IkB is phosphorylated and then degraded via ubiquitin pathway. Free dimer of Nf-κB is released and transferred to nucleus. NF-κB induced series of expressions of genes such as FLIP, cIAP1 and cIA2 etc. and the products of expressions inhibit apoptosis. Apoptosis inhibitor XIAP (X-chromosome-linked IAP) is also mediated by NF-κB. XIAP inhibits caspase-3 and caspase-7 by binding to the end of NH2 with its BIR domain, thus inhibits apoptosis.

  • Intracellular factors-induced apoptosis:

Intracellular factors induced apoptosis is the major cell death pathway in vertebrate. It can be activated by various factors such as retreat of growth factor, activation of heat shock genes, DNA damage, reactive oxygen, excessive intracellular calcium ion and other stress reactions. These factors result enhanced permeability of mitochondrial outer-membrane and release of apoptosis-inducing proteins such as apoptosis inducing factor (AIF), cytochrome C, Smac/DIABIO etc. The release of cytochrome C from the mitochondria doesn’t depend on decline of mitochondrial surface potential or enhancement of the permeability of outer-membrane. Smac /DIABIO is an inhibitor of apoptosis-inhibitor protein XIAP. AIF is independent of caspase pathway, and induce apoptosis, too. It is transferred into nucleus after entering in cytoplasm. Probably with endonuclease G, AIF induces condensation of chromatin and fragmentation of high-molecular weight DNA (50kb).

It is key event in apoptosis that cytochrome C is released from intracellular mitochondria. Once cytochrome C is released to cytoplasm, it binds to Apaf-1 molecule which is an apoptosis activator, then they recruit precursor of caspase-9 to form a multi-protein complex called apoptosome. Activated caspae-9 then leads to downstream activation of caspase-3 and caspase-7.

The increase of permeability of mitochondrial outer-membrane is due to the changes of permeability transition pore (PTP) or activation of apoptosis activator in Bcl-2 family. PTP complex consists of voltage dependent anion channel on mitochondrial outer-membrane, adenine nucleotide transferase on mitochondrial inner-membrane and cyclophilin D in mitochondrial matrix. High level calcium ion induces the open of the PTP which then leads to diffusion of water and 1.5 kD solute molecule from cytoplasm to mitochondrial matrix, then causes expansion of mitochondria and disintegration of the trans-membrane potential. The other mechanism that induces the increase of the permeability of mitochondrial outer-membrane refers to Bcl-2 family.

There are two groups of proteins in Bcl-2 family: Bcl-2 and Bcl-XL are apoptosis inhibitors; Bad, Bax and Bid are apoptosis-inducing proteins. Cells’ susceptibility to apoptosis depends on the balance of the two groups of proteins. When apoptosis-inducing proteins are more, cells tend to be susceptible to apoptosis. On the contrast, when apoptosis inhibitors are dominant, cells tend to have resistance. Excessive Bcl-2 on mitochondrial surface is thought to be the key point of PTP’s formation.

Apoptosis-inducing proteins of Bcl-2 family spread all over the cytoplasm and are sensors of any cell damage or stress. When cell stress occurs, they transfer to locations of apoptosis inhibitors on mitochondrial surface. Their interactions disturb the functions of apoptosis inhibitors and leads to the formation of PTP complex and release of other molecules. Then apoptosome is generated and cascade reactions of caspases are activated.

Bid is the linkage of death receptors and mitochondria pathway. Bid exists in cytoplasm with an unactivated state, and transfers to mitochondrial membranes after being activated. Then Bid antagonizes the apoptosis inhibitors of Bcl-2 to induce apoptosis. In some types of cells, Bid is cut into activated tBid by caspase-8 after being activated by death receptors. tBid then binds to the mitochondrial membranes and interacts with Bax, making Bax inserted in the membranes and oligomerized. Mitochondrial membranes turn more permeable, and cells get into apoptosis program. Bcl-XL can inhibit the combination of tBid and Bax and thus inhibit apoptosis. Moreover, Bid can be activated by other enzymes such as granzyme B and protease in lysosome.

Executants in apoptosis process:

Caspases are major executants in apoptosis. They are cysteine protease with presence of inactive state in cells. During apoptosis these pro-enzymes are cut into active enzymes. Extracellular factors induced apoptosis leads to activation of caspase-8 or caspase-10. These caspases activate other caspases in cascade reactions. The cascade reactions eventually lead to activation of effective caspases such as caspase-3 and caspase-6. Effective caspases cleave important intracellular proteins like cytoskeletal proteins and causes morphologically changes of cells.

Caspase-3 is considered to be the most important executant. It is activated by upstream caspases (caspase-8, caspase-9 or caspase-10). Caspase-3 specifically activates endonuclease CAD (caspase activated DNase). In proliferating cells, CAD normally combines with ICAD (an inhibitor of CAD) to form an inactive complex. In apoptosis, ICAD is cut by caspase-3 and release CAD, followed by rapid fragmentation of DNA.

Other than death receptors, there are other mechanisms that can activate cascade reactions of caspases. Granzyme B can be transported into cells by cytotoxic T lymphocyte and directly activate caspase-3, caspase-7, caspase-8 and caspase-10. Mitochondria is important regulator of caspase-cascade reactions and apoptosis. Released cytochrome C from mitochondria leads to activation of caspase-9, and caspase-3. This process is mediated by formations of apoptotic bodies.

PARP (Poly Adp Ribose Polymerase) is the first substrate of caspases that has been identified. PARP catalyzes production of ADP-ribose via which it binds to DNA cracks and modifies nuclear proteins, and it means PARP participates in the repairment of DNA damage. Caspase-3 can cut PARP and stop its repairing function. Lamin is a nuclear protein that is capable of sustaining shapes of nucleus and regulating interactions of chromatin and nuclear membranes. The degradation of lamin by caspase-6 will lead to condensation of chromatin and fragmentation of nucleus.

In spite always we focus on apoptosis and caspases, they can’t represent everything in field of apoptosis. There are other enzymes that are important in executing programed cell death including calpain, cathepsin, endonuclease and some other protease. They function alone or cooperatively with assistance of some organelles like mitochondria, lysosome and ER. More attentions should be paid on factors other than caspases in order to find effective therapeutic solutions for curing diseases like cancer.

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