Apoptosis Discovery Carl Vogt, German Scientist Was The First To

APOPTOSIS DISCOVERY Carl Vogt, German scientist was the first to describe its principle in 1842. Later,Kerr,Wylie,currie suggested the term apoptosis and they were the first to use this term.

DEFINITION Apoptosis is programmed cell death. Derived from Greek ‘’falling off’’. It’s completely normal physiological, energy dependent orderly process required for the maintenance of normal haemostasis. Apoptosis takes place during fetal development (finger and toe dev), removal of endometrium (menstrual cycle), formation of synapses in brain (loss of surplus cells).Bcl-2,Bax ,paf-1,Caspases biochemical features of apoptosis •

Protein cleavage by caspases

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DNA breakdown- by endonucleases triggered by caspase activity.

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Phagocytic recognition- by expression of surface markers on the apoptotic cell.

HISTORY STEPS A intrinsic property of normal cell metabolism.

1. Shrink. 2. Develop bubble-like blebs on their surface. 3. Chromatin (DNA + protein) begins to degrade. 4. Mitochondria break up releasing cytochrome c 5. Cells break into small membrane wrapped fragment 6. The phospholipid, phosphatidylserine is exposed on surface 7. Cell fragments are removed by phagocytosis. MECHANISMS OF APOPTOSIS 4 Pathways 1. -Extrinsic (death receptor mediated pathway) This pathway is typically engaged in the immune system and is the method used to delete activated T-cells at the end of an immune response. This is mainly perforin/granzyme mediated The best characterized death receptors comprise CD95 (APO-1/Fas), TNF receptor 1 (TNFRI), TRAIL-R1 and TRAIL-R2, while the role of DR (TRAMP/Apo-3/WSL-1/LARD) orDR6 has not exactly been defined. Extrinsic pathway involve signals such as the binding of death inducing ligands to cell surface receptors called death receptors. These ligands can either be soluble factors or can be expressed on the surface of cells.

This pathway is triggered by the death factors e.g. TNF, Fas (CD95) binding with the death-receptor superfamily e.g. Fas and TNFR-1.

Binding to the receptor induces receptors to cluster and trimerise.

FADD (Fas associated death domain protein) is recruited via its death domains The DED (death effector domain) of FADD recruits pro-caspase 8 via its DED The complex brings multiple pro-caspase 8 molecules in close proximity, leading to their activation through ‘induced proximity’ (the aggregation of pro-caspase 8 molecules results in their cross-activation). This is the DISC. Activated caspase-8 (a heterotetramer) is releasedfrom DISC into the cytoplasm where it functions as an initiator caspase, activating downstream executioner caspase, primarily via procaspase-3.

2. -Intrinsic (Mitochondrial pathway) • This pathway is usually activated in response to other lethal stimuli such as DNA damage, oxidative stress and hypoxia . Mitochondria contain pro-apoptotic factors such as cytochrome c, AIF (apoptosis inducing factors), Smac/DIABLO, Omi/HtrA2 endonuclease G, caspase-2 , caspase-9 from the mitochondrial intermembrane space 1. -Interaction b/w activated CTL receptor and MHC-class 1 molecule 2. -Integrin mediated death Causes of Apoptosis 1. Morphology of cell changes mainly due to the action of proteases. These are cysteine proteases which cleave proteins at aspartic acid residues. 2. These are known as caspases (Cysteine Aspartate Specific ProteASEs). 3. Proteolytic cleavage of the caspases at conserved aspartic acid residues activates enzymes of 10 and 20kDa subunits. 4. Normally, these proteins are present as inactive zymogens in all cells. 5. Active caspases are hetero dimers with 2 large 20Kda and 2 small 10 Kda subunits and 2 active sites.

Death receptor mediated pathway

MISREGULATION OF APOPTOSIS APOPTOSIS AND CANCER EXPERIMENTAL ASSAY DECTION OF APOPTOSIS INVITRO

Apoptotic signalling from the death receptors Binding of the death inducing ligand (Fas ligand, TNF α and TRAIL), to its receptor can lead to the generation of ceramide. Ceramide release promotes lipid raft fusion resulting in clustering of the death receptors which is required to amplify signalling. Following ligand binding a conformational change in the intracellular domains of the receptors reveals the presence of a “death domain” which allows the recruitment of various apoptotic proteins to the receptor. The protein complex is often called the DISC (death inducing signalling cascade).

The final step in this process is the recruitment of one of the caspases, typically caspase 8, to the DISC. This results in the activation of caspase 8 and the initiation of apoptosis.

TNF (Tumour necrosis factor) receptor signalling TNF is produced by T-cells and activated macrophages in response to infection. By activating its receptor (TNFR1), TNF can have several effects. TNF can induce apoptosis, although receptor ligation is not enough on its own to initiate apoptosis as is the case with Fas ligand binding. Binding of TNFα to TNFR1 results in receptor trimerisation and clustering of intracellular death domains. This allows binding of an intracellular adapter molecule called TRADD (TNFR1-associated death domain), via interactions between death domains. TRADD has the ability to recruit a number of different proteins to the activated receptor. Recruitment of TRAF2 (TNF-associated factor-2), can lead to activation of NF-kB and the JNK pathway. TRADD can also associate with FADD, which leads to the induction of apoptosis via the recruitment and cleavage of pro-caspase 8. Signalling by Fas (CD95) The ligand for Fas, FasL or CD95, activates apoptosis in a similar way to the TNF receptor. Binding of the ligand promotes receptor clustering, DISC formation and the activation of the caspase cascade. The adaptor protein FADD can be recruited directly to the death domain on the fas receptor, without requiring the prior recruitment of TRADD. The Fas receptor is thought to only activate apoptosis and does not play an important role in other aspects of cell signalling like the TNF receptor Induction of apoptosis by TRAIL In a number of ways TRAIL, (TNF-related apoptosis inducing ligand), is similar to FasL. Binding of TRAIL to its receptors DR4 and DR5 triggers rapid apoptosis in many cells. There are also decoy receptors that compete for binding of TRAIL with DR4 and DR5 receptors. The decoy receptors are called DcR1 and DcR2. Both of these receptors are capable of competing with DR4 and DR5 receptors for ligation, however, binding does not initiate apoptosis since DcR 1 does not posses a cytoplasmic domain, while DcR2 has a truncated death domain lacking 4 out of the 6 amino acids essential for recruiting adaptor proteins. Induction of apoptosis by TRAIL

Intrinsic pathway are produced following cellular stress. Cellular stress may occur from exposure to radiation or chemicals or to viral infection. In general intrinsic signals initiate apoptosis via the involvement of the mitochondria. ratios of the various bcl-2 proteins can often determine how much cellular stress is necessary to induce apoptosis. Role of Bcl-2 proteins Bcl-2 proteins play a pivotal role in regulation of the mitochondrial pathway. Comprised of pro apoptotic molecules: Bax, Bek, Bad ......and anti-apoptotic molecules: BCl2, Bcl-X and Mcl-1. Upon apoptosis induction – pro-apoptotic BCl-2 proteins with multidomains i.e Bax, translocate into the mitochondria and form a pore like structure by oligomerization. They promote cytochrome C release. The release of cytochrome c from the mitochondria is a particularly important event in the induction of apoptosis. Once cytochrome C has been released into the cytosol it is able to interact with a protein called Apaf-1. This leads to the recruitment of pro-caspase 9 into a multi-protein complex with cytochrome C and Apaf-1 called the apoptosome. Formation of the apoptosome leads to activation of caspase 9 and the induction of apoptosis. Translocation into the mitochondria is dependent on proteins which contain a BH3 domain. Anti-apoptotic molecules exert their effect by sequestering BH3 domain only proteins in mitochondrial complexes, preventing their activation or translocation. Caspases and apoptosis Caspases are a family of proteins that are one of the main executors of the apoptotic process. They belong to a group of enzymes known as cysteine proteases and exist within the cell as inactive pro-forms or zymogens. These zymogens can be cleaved to form active enzymes following the induction of apoptosis. Induction of apoptosis via death receptors typically results in the activation of an initiator caspase such as caspase 8 or caspase 10. These caspases can then activate other caspases in a cascade. This cascade eventually leads to the activation of the effector caspases, such as caspase 3 and caspase 6. These caspases are responsible for the cleavage of the key cellular proteins, such as cytoskeletal proteins, that leads to the typical morphological changes observed in cells undergoing apoptosis. Apoptosis and cancer Regulation of death receptor signalling in cancer

Somatic mutations of TRAIL receptors DR4 and DR5 have been found in some B-cell nonHodgkin lymphomas (BNHL). These mutations target the death domain resulting in malfunction of apoptotic signal transduction. Caspases Mutations have been found in some tumours including colorectal cancer as well as head and neck carcinomas. It is believed that these caner cells have impaired caspase expression and function caused by epigenetic mechanisms such as gene silencing. Caspase-8 expression has been found to be inactivated by hypermethylation in various tumour cells, although other gene inactivation mechanisms may also be involved. Mutations of Caspase-10 and Fas have lead to the inactivation of death effector domain which is necessary for the caspase-10/FADD interaction in the DISC. This has been indentified in 15% of BNHL patients. Translocations involving the BCL-2 gene are the hallmark of follicular lymphoma. The translocations causes BCL-2 deregulated expression by placing BCL-2 under the control of IgHµ enhancer, resulting in high levels of Bcl-2 protein. The translocated BCL-2 can accumulate somatic point mutations. These mutations may contribute to deregulation of BCL-2 gene expressions or alter the function of the protein. The translocation t(1;14)(p22; q32) is associated with MALT- lymphoma affecting the BCL10 gene. This results in deregulation expression of the gene. Apoptosis pathways in cancer and cancer therapy Activation of apoptotic pathways is a key mechanism by which cytotoxic drugs kill tumour cells. Defects in apoptosis signalling contribute to resistance of tumours. Cytotoxic drugs activate the mitochondrial, intrinsic, pathway of apoptosis. Death receptor extrinsic pathways contributes to sensitivity of tumour cells towards cytotoxic treatment. Pro-apoptotic signaling in cancer therapy: Caspases Caspases are effector molecules in various forms of cell death. The ability of anticancer agents to trigger caspase activation appears to be a critical determinant of sensitivity or resistance to cytotoxic therapies. As a consequence inhibition of caspase activation may be an important factor in chemoresistance.

Pro-apoptotic signalling in cancer therapy: Caspases Caspase-8 expression was found to be frequently inactivated by hypermethylation of regulatory sequences of the caspase 8 gene in tumour cells from neuroblastoma. Restoration of caspase 8 expression by gene transfer or demethylation treatment, sensitized resistant tumour cells to death receptor induced or drug induced apoptosis. Anti-apoptotic signalling in cancer therapy: Bcl-2 proteins Mutations or altered expression of pro/anti apoptotic molecules can drastically alter drug response in experimental systems. Clinical correlative studies have shown that high level expression of anti-apoptotic Bcl-2 confers a clinically important chemoresistant phenotype on cancer cells. Likewise, reduced BAX levels are associated with poor responses to chemotherapy and shorter overall survival in breast/colon carcinoma. Inhibitor of apoptosis proteins (IAPS) IAPS directly inhibit activated caspases. In addition to regulation of apoptosis, IAP members such as survivin, are involved in the regulation of mitosis. IAP activity is stimulated in part by transcription factor NFкB and negatively regulated by caspase mediated cleavage. In addition, Smac/Diablo and Omi, two proteins released from the mitochondria upon apoptosis induction , neutralize IAPS through binding to them and displacing them. IAP inhibitor Smac/Diablo IAPS cause inhibition of drug induced apoptosis and high IAP expression correlates with poor treatment response and adverse prognosis. The IAP inhibitor Smac/Diablo has prompted interest: expressing Smac/Diablo in the erythroplasma of tumor cells may overcome IAP mediated inhibition of apoptosis induction in tumours.