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Apo: Greek: from, away Ptosis: Greek: fall, falling A specific type of cell death first described in 1972 by Kerr, et al in the British Journal of Cancer Programmed Cell Death (Lockshin and Williams, 1964) Developmental Event, may or may not be apoptotic
In all our tissues, the size of individual cell populations is governed by cell birth brought about by division of cells - mitosis - and by a controlled cell deletion or 'suicide' process known as apoptosis. The name apoptosis is derived from the ancient Greek term meaning 'the falling of leaves from trees or petals from flowers'. During embryonic development, we are fashioned not only through mitosis, cell differentiation and cell migration, but also through the 'useful cell death' that is apoptosis. Numerous tissues within our adult bodies are maintained by a fine balance between mitosis and apoptosis. For example, immune responses to infection are boosted by cell division and finely tuned and curtailed by apoptosis; cells of the gut and skin are renewed through cycles of mitosis and apoptosis; the lactating breast regresses post-lactation by apoptosis.
Apoptotic B Cells
Essential for proper development of the multicellular organism "Webbed" tissue between the digits of developing human embryos is removed by apoptosis Tadpole tail removed by apoptosis during development
Neuronal cell death by apoptosis is fundamental for CNS development
Skin cells (keratinocytes) undergo apoptosis and migrate to the surface where they form the protective outer layer of skin
Autoreactive T cells with the potential to attack "self" are removed by apoptosis At the termination of an immune response when they are no longer needed
Necrosis
Apoptosis
Morphological changes
Loss of membrane integrity. Begins with swelling of cytoplasm and mitochondria. No vesicle formation, complete lysis Membrane blebbing, but no loss of integrity. Aggregation of chromatin at nuclear memb. Begins with shrinking of cytoplasm and condensation of nucleus. Formation of membrane bound vesicles (apoptotic bodies)
Biochemical changes
No energy requirement passive process Random digestion of DNA Postlytic DNA fragmentation Energy (ATP)-dependent Non-random mono- and oligonucleosomal length fragmentation of DNA. Laddering pattern Prelytic DNA fragmentation
Physiological changes
Phagocytosis by macrophages Phagocytosis by adjacent cells or macrophages
No inflammatory response
Role in Diseases:
Over-active apoptosis: examples are neurodegenerative diseases (eg Alzheimer's), immunodeficiency diseases (eg. AIDS), Stroke and coronary heart disease. Under-active apoptosis: examples are cancer, auto-immune and chronic inflammatory diseases (eg rheumatatoid arthritis). The latter may be caused by defects in the mechanisms of apoptotic-cell clearance.
Apoptosis is activated through two principal signaling pathways: intrinsic and extrinsic
Cancer is often initiated by DNA damage Normal cells undergo apoptosis in response to stress-inducing events in the cell, such as DNA damage Dysregulation of apoptosis is critical for cancer development and tumor cell survival
Sydney Brenner of the Salk Institute used the nematode Caenorhabditis elegans, which became a multicellular model experimental system, to follow cell division and differentiation from the fertilized egg to the adult via microscopic observation. He demonstrated that a specific gene mutation, induced by ethyl methane sulfonate, could be linked to a specific effect in nematode organ development. His work on nematodes created an experimental system that laid the foundation for the study of apoptosis.
John Sulston of the Wellcome Trust Institute in England mapped cell lineages, where every cell division and differentiation could be followed in the development of C. elegans. There are only 959 cells in an adult nematode. He showed that specific cells lineages (nerves) undergo programmed cell death, as an integral part of the normal differentiation process. Robert Horvitz of MIT discovered and characterized key genes controlling cell death in C. elegans. He identified the first two bona fide "death genes", ced-3 and ced-4. Functional ced-3 & ced-4 genes are a prerequisite for cell death to be executed. Another gene, ced-9, protects against cell death by interacting with ced-3 and ced-4. He has shown how these genes interact with each other in the cell death process and that corresponding genes (a ced-3-like gene) exists in humans.
The C. elegans genome is 9.7 x 107 bp and is now fully sequenced (24 x E. coli, 1/38 of human). The 19099 genes include 790 seven-pass transmembrane receptors, 480 zinc finger proteins, and 410 protein kinases; see News and Views, Nature 396: 620621 (1998). The life cycle of C. elegans from egg to sexual maturity (and new eggs) is about 3 days. The adult hermaphrodite consists of exactly 959 somatic cells of precisely determined lineage and function. Individual cells are named and their relationships to their neighbours are known.
Overall the 959 cells of adult C.elegans arise from 1090 original cells; exactly 131 cells undergo programmed cell death in the wild type worm. Of the 1090, 302 are neurons, and many of the programmed deaths also lie in the neuronal lineage.
Molecular nature of the PCD came from genetic studies done on C.elegans.
131 cells out of 1090 cells undergo PCD: genetic screens of mutants identified Egl-1, CED3, CED4 and CED 9 to be involved. Egl-1, CED3, CED4 are death promoters since their loss of function results in survival of all 131 doomed cells. CED 9 is death inhibitor since its loss of function causes embryonic lethality by massive ectopic cell deaths.
Fundamental components of apoptotic pathways are conserved across the species. Ced -9 is similar to human oncogene :Bcl2. Ced-3 has a mammalian counterpart, originally known as ICE (Interleukin 1 Converting Enzyme), now termed Caspase 1. Ced-4 acts as an adapter for caspase activation; the mammalian counterpart Apoptosis activating factor Apaf-1.
Expressed widely in cells in inactive proenzyme form (pro-caspase) Must be activated for proteolysis
Responsible for the more visible "hallmarks" of apoptosis More Notable Members Caspases 3, 6, 7: Important downstream effector caspases Caspase 8: initiator in death receptor pathway Caspase 9: Initiator in intrinsic pathway. Activated by conformational change
Effector Caspases: Activate downstream caspases and act on Various cellular substrates
In the first example, the effector caspases cleave an inhibitor or an effector protein. An example of this would be CAD (Caspase-Activated Deoxyribonuclease), and ICAD (Inhibitor of CAD). When ICAD binds to CAD, CAD is kept inactivated. However, active effector caspases cleave ICAD which then releases CAD. CAD can then cleave the DNA into fragments (forming the characteristic DNA laddering of apoptotic cells). The second example illustrates that the effector caspases can also cleave structural proteins, such as the nuclear lamins. Nuclear lamins maintain the integrity of the nucleus, but when they are cleaved by the effector caspases, the nucleus condenses (another characteristic of apoptotic cells). Finally, in the third example, effector caspases can cleave off the auto-inhibitory domains of certain proteins. A good example of this would be PAK2. When the effector caspase cleaves off the auto-inhibitory domain of PAK2, PAK2 now becomes constitutively active, playing a role in the membrane blebbing that is characteristic of apoptotic cells.
BCL-2
BCL-2 is a human proto-oncogene located on chromosome 18. Its product is an integral membrane protein (called Bcl-2) located in the membranes of the endoplasmic reticulum (ER), nuclear envelope, and in the outer membranes of the mitochondria.
The gene was discovered as the translocated locus in a B-cell lymphoma
In the cancerous B cells, the portion of chromosome 18 containing the BCL-2 locus has undergone a reciprocal translocation with the portion of chromosome 14 containing the antibody heavy chain locus. This t(14;18) translocation places the BCL-2 gene close to the heavy chain gene enhancer.
Bcl-2 Members
Bcl-2 :first protooncogene gene to be discovered from the family: cloned from t(14;18) breakpoint in follicular lymphoma
BH1 and BH2: in death antagonists, allow heterodimerization with Bax to repress apoptosis
BH3: in death agonists (eg. Bax, Bak) allows heterodimerization with Bcl-XL and Bcl-2 to promote apoptosis
BH4: conserved in apoptosis antagonist members (eg. Bcl-XL) but absent in apoptosis agonists (except Bcl-Xs), this domain allows interaction with death regulatory proteins such as Raf-1, Bad, and perhaps Ced-4.
Their hydrophobic carboxy-terminal domain helps target them to the cytoplasmic face of three intracellular membranes.
Bcl2 is an integral membrane protein, even in healthy cells, whereas Bcl-w and Bcl-xL only become tightly associated with the membrane after a cytotoxic signal. A hydrophobic groove, formed by residues from BH1, BH2 and BH3, can bind the BH3 -helix of an interacting BH3-only relative.
They are thought to act by binding to and neutralizing their pro-survival relatives.
They act upstream of Bax sub-family of proteins.
Bcl-2 family members can regulate apoptosis related mitochondrial changes such as the permeability transition pore (PT)
The PT pore is a poly protein channel, comprising of VDAC and PBR on the outer membrane and ANT and cychlophilin D on the inner membrane. Cytochrome c release may occur through outer membrane rupture resulting from mitochondrial swelling caused by PT pore perturbation.
Bcl-2, Bcl-xL and Bax have been shown to form ion channels in synthetic lipid membranes.
Thus, Bcl-2 members having the BH1 and BH2 domains may function by forming pores in organelles such as mitochondria or rather, stabilize or perturb the pre-existing channel, PT.
Involves closure of voltage dependent ion channel (VDAC) and impairment of ATP-ADP exchange. Opening of permeability transition pore (PTP).
The Role of Mitochondria in Apoptosis The mitochondrion has been identified as playing a central role in apoptosis 1. Bcl-2 and Bcl-XL localize to the mitochondrial membrane.. 2. Bcl-2 can recruit kinases such as Raf-1 which are involved in mediating Bcl-2's death antagonizing action. 3. Bcl-2/-XL can recruit ced-4 and its mammalian homolg, Apaf-1 to the mitochondrial membrane. This may prevent ced-4/Apaf-1 from activating caspases, thereby inhibiting apoptosis. 4. Mitochondial proteins, when leaked into the cytosol, are capable of inducing apoptosis. During apoptosis, cytochrome c and SMAC are released from the mitochonria and with other factors, such as Apaf-1 (apoptosis protease activating factor-1) and Apaf-3, lead to caspase activation and apoptosis. Increased levels of Bcl-2 can prevent the release of these molecules, whereas, caspase inhibitors cannot. This indicates the release of cytochrome c and SMAC is downstream of Bcl-2 function but upstream of the caspases. 5. Apoptosis is associated with a change in the mitochondrial membrane potential, a phenomenon known as permeability transition (PT). The PT can be blocked by excess Bcl-2 but not by inhibitors of caspases, indicating the PT is downstream of Bcl-2 but upstream of caspase activation. 6. Bcl-2, Bcl-XL, and Bax are capable of forming selective ion pores in membranes. Thus, they may form channels in the mitochondrial membrane that could regulate the PT and the release of molecules such as cytochrome c and AIF.
The BAX gene, the promoter of apoptosis, is mutated in genetically unstable cancers of the colorectum, stomach, and endometrium (1998)
Inactivating mutation of the pro-apoptotic gene BID in gastric cancer (2004) (6%)
(4.3%) 2004
Caspase-8 gene is frequently inactivated by the frameshift somatic mutation 1225_1226delTG in hepatocellular carcinomas (2005) 10%
Receptors are Type I transmembrane proteins: intracellular C terminal tail, membrane spanning region, an extracellular ligand binding domain.
Significant homology in 60-80 aa cytopl. sequence Death domain (DD). Death receptors are activated by their natural ligands: group of cytokines belonging to TNF family.
Role of Fas/FasL Deletion of activated T-cells at the termination of an immune response Cytotoxic T-cell mediated killing of cells (virus-infected, cancerous) Destruction of inflammatory or immune cells in immuneprivileged sites (i.e, eyes, reproductive organs)
Pathways to Apoptosis
The IAP proteins have been divided into three classes (classes 1, 2, and 3) based on the presence or absence of a RING finger and the homology of their BIR domains.
IAPs are characterized by 70-80 aa Baculoviral IAP Repeat (BIR) domains. IAPs with multiple BIR domains use third BIR domain to inhibit caspase 9 and second BIR domain to inhibit caspase 3 ,7. BIR 1 domain has no caspase inhibiting activity and is least conserved. RING domain function as adaptors : provide specificity for proteosomal degradation. NOD domain facilitates self association whereas coiled coil domain mediates interaction with Beta Tubulin.
IAP regulators
Regulatory IAP-binding proteins were first identified in Drosophila. The proteins Reaper, Hid, Grim, and Sickle were shown to bind and inhibit the Drosophila IAP, DIAP1. Later, human IAP inhibitors identified called SMAC/DIABLO and Omi/HTRA2. These IAP inhibitors share a homologous sequence in their NH2 terminus that is responsible for binding and inhibiting IAPs.
HTRA2 binds to the BIR3 domain of XIAP, but with weaker affinity than SMAC. In addition to inhibiting IAPs through binding the BIR3 pocket, HTRA2 can also cleave and inactivate multiple IAPs including XIAP,cIAP1, and cIAP2, but not survivin. Omi has serine protease activity
Caspase-independent Apoptosis
Death associated with activation of lysosomal and proteosomal Proteases and granzyme B and matrix metalloproteases, calpains. Many programmed or physiological deaths do not appear to depend on caspase activation.
Pyruvate + NADH
NADH exhibits fluorescence at an excitation wavelength of 360 nm with emission at 450nm. Velocity of decrease of Ex 360nm/Em450 nm indicates conversion of NADH to NAD and hence activity of LDH.
3) Clonogenic assay: Ability of cells to divide and form colonies is called clonogenic activit
However, integrity of pl memb is not compromised till late stage and hence not very usefu
In normal cells, phosphoidylcholine and sphingomyelin are on external leaf and phosphotidylethnolamine and phosphatidylserine (PS) are in the inner leaflet . Redistribution of phospholipids in the plasma memb. is an early apoptotic change. Annexin V conjugated with flourophore like FITC can bind to exposed to PS in Ca dependent manner. Can be viewed microscopically or by flow cytometry.
Annexin V staining
Western blot with antibodies specific to individual members. If subcellular localisation is not relevant: cells can be lysed in non-ionic detergent. If subcellular is needed, cellular components are subfractionated to obtain mitochondrial and cytosolic fractions. To determine Bax or Bak oligomers, crosslinking agents are added e.g disuccinimidyl suberate (DSS), Bis (Sulphosuccinimidyl) suberate (BS3) to the mitochondrial fraction suspended in isotonic buffer . The crosslinker is quenched with 1 M Tris-HCl ph7.5. Membranes are then lysed in radioimmunol precipitation assay (RIPA) buffer and cleared by centrifugation at 12000g: analysis by SDS-PAGE and Western Blot. Oligomerisation is visualised using Western blot as a high molecular weight species.
Probes can be directly added to cultured cells or isolated mitochondria, incubated for 15 min and harvested to be analyzed by flow or fluorescent microscopy.
Nuclear condensation and fragmentation can be visualised by staining with fluorescent dyes e.g Hoechst (bisbenzimide) and DAPI (46diamidino 2-phenylindole, dilactate).
Intensity of staining in the nucleus is proportional to the extent of apoptosis due to increased permeability of the dyes.
Tamoxifen Treated
Tamoxifen
G1
G2
G0
Degradation of nuclear DNA results in decrease in DNA content or hypoploidy. Cells are suspended in ice cold PBS, fixed with cold ethanol. Cells are permeabilised with Triton x100, stained with PI and analysed with flow. However, necrotic and other cellular debris can also get included.
DNA Fragmentation:
Cleavage of DNA at nucleosomal sites results in 180-200 bp fragments which appears as DNA ladder. Quantitative measurement : amount of fragmented DNA is proportional to frequency of apoptosis.
Based on the principle that TdT mediates incorporation of biotinylated dUTP into 3 OH ends of fragmented DNA .
Cells are permeabilised using Triton X 100 or Proteinase K in case of formalin fixed tissue sections.
After wash, cells /Tissues can be incubated either with FITC conjugated avidin for detection under fluoroscent microscope or avidin-HRP conjugate can be added followed by DAB.
Preclinical Preclinical Preclinical Preclinical Clinical Preclinical Preclinical Preclinical Clinical Preclinical Preclinical Preclinical Preclinical Preclinical Preclinical
IFI-983L, IFI-194 Small molecule CPM-1285 analogs Lipidated peptide Terphenyl derivative Peptidomimetic SAHBs Stapled peptide
4-PhenylsulfanylphenylamineDerivatives Small molecule Proapoptotic inhibition (BID) The Burnham Institute 3,6-Dibromocarbazole Piperazine derivatives of 2-propanol Small molecule Proapoptotic inhibition (BAX) Serono Humanin peptides Peptide Proapoptotic inhibition (BAX) The Burnham Institute Ku70 peptides Peptide Proapoptotic inhibition (BAX) The Blood Center of South Eastern Wisconsin
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