APOPTOSIS In the human body about 100,000 cells are produced every second by mitosis and a similar number die by apoptosis !!! There is a nuclear enzyme involved in DNA repair called Poly ADP-ribose polymerase, PARP. In apoptosis, this enzyme is split by an ICE(Interleukin converting enzyme)-like protease. A product from Boehringer Mannheim, Anti-PARP, detects these PARP fragments in early apoptotic cells.

Definition Apoptosis is a peculiar well controlled individual cell death that is caspase mediated and leads to fragmentation of the cell and organelles into numerous small buds, which are then engulfed by macrophages without surrounding inflammation. Apoptosis can be defined as 'gene-directed cellular self-destruction'; it is sometimes referred to as 'programmed cell death' although this is really a phenomenon where cells are programmed to die at a particular point, e.g. during embryonic development, and even here cells may go through an apoptotic pathway. However, apoptosis is certainly a distinct process from other forms of oncosis leading to necrosis.

Importance of Apoptosis 1) Crucial for embryonic development -Errors in Apoptosis can lead to Birth Defects 2) Important for maintaining homeostasis - Cell death is balanced with mitosis to regulate cell number. 3) Improper regulation contributes to human disease - Neurodegenerative diseases Parkinson’s Alzheimer’s - Cancer - Autoimmune diseases e.g. (diabetes type I) - Viral diseases Apoptosis, or programmed cell death, is a normal component of the development and health of multicellular organisms. Cells die in response to a variety of stimuli and during apoptosis they do so in a controlled, regulated fashion. This makes apoptosis distinct from another form of cell death called necrosis in which uncontrolled cell death leads to lysis of cells, inflammatory responses and, potentially, to serious health problems. Apoptosis, by contrast, is a process in which cells play an active role in their own death (which is why apoptosis is often referred to as cell suicide). Upon receiving specific signals instructing the cells to undergo apoptosis a number of distinctive changes occur in the cell. A family of proteins known as caspases are typically activated in the early stages of apoptosis. These proteins breakdown or cleave key cellular components that are required for normal cellular function including structural proteins in the cytoskeleton and nuclear proteins such as DNA repair enzymes. The caspases can also activate other degradative enzymes such as DNases, which begin to cleave the DNA in the nucleus.

Characteristics It is a process that occurs in almost all living creatures since their early stages of embryological development. It is an active cytological process in which energy is consumed (ATP dependent) It is programmed or controlled by genetic protocol or program (control of enzymes, cell membrane surface proteins & cytoplasmic molecules, signal transduction, gene expression) It may be triggered by intrinsic or extrinsic stimuli Typically, the cell begins to shrink following the cleavage of lamins and actin filaments in the cytoskeleton (A). The breakdown of chromatin in the nucleus often leads to nuclear condensation and in many cases the nuclei of apoptotic cells take on a "horse-shoe" like appearance (B). Cells continue to shrink (C), packaging themselves into a form that allows for their removal by macrophages. These phagocytic cells are responsible for clearing the apoptotic cells from tissues in a clean and tidy fashion that avoids many of the problems associated with necrotic cell death. In order to promote their phagocytosis by macrophages, apoptotic cells often ungergo plasma membrane changes that trigger the macrophage response. One such change is the translocation of phosphatidylserine from the inside of the cell to the outer surface. The end stages of apoptosis are often characterised by the appearance of membrane blebs (D) or blisters process. Small vesicles called apoptotic bodies are also sometimes observed (D, arrow).

Morphology Cell shrinkage (condensation of cytoplasm) due to breakdown of cytoskeleton Breakdown of mitochondria; release of cytochrome C Nuclear condensation Nuclear fragmentation Cell membrane blebbing Fragmentation; apoptotic body formation: membrane-bound cellular fragments, which often lack nuclei Phagocytosis There are a number of mechanisms through which apoptosis can be induced in cells. The sensitivity of cells to any of these stimuli can vary depending on a number of factors such as the expression of pro- and anti-apoptotic proteins (eg. the Bcl-2 proteins or the Inhibitor of Apoptosis Proteins), the severity of the stimulus and the stage of the cell cycle. Some of the major stimuli that can induce apoptosis are outlined in the illustration below.

Cellular changes associated with apoptosis In some cases the apoptotic stimuli comprise extrinsic 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 such as cytotoxic T lymphocytes. The latter occurs when T-cells recognise damaged or virus infected cells and initiate apoptosis in order to prevent damaged cells from becoming neoplastic (cancerous) or virus-infected cells from spreading the infection. Apoptosis can also be induced by cytotoxic T-lymphocytes using the enzyme granzyme. In other cases apoptosis can be initiated following intrinsic signals that are produced following cellular stress. Cellular stress may occur from exposure to radiation or chemicals or to viral infection. It might also be a consequence of growth factor deprivation or oxidative stress caused by free radicals. In general intrinsic signals initiate apoptosis via the involvement of the mitochondria. The relative ratios of the various bcl-2 proteins can often determine how much cellular stress is necessary to induce apoptosis.

How Apoptosis Differs from Necrosis? Apoptosis is intrinsically controlled, necrosis is not Apoptosis is more rapid (12-24 hours) than necrosis Apoptosis is induced by endogenous or exogenous stimuli, necrosis is always induced by exogenous harms Apoptosis is limited to single or few cells at a time, and occurs among healthy cell population, necrosis is usually more extensive & occurs in tissue exposed to injuries Cell cytoplasm shrinks in apoptosis and swells in necrosis. Nucleosomes of apoptotic cells are 180 bp fragments, contrary to the irregular ones in necrosis Apoptosis has no inflammation, while necrosis leads to liberation of pro-inflammatory mediators Apoptosis has no systemic manifestations contrary to most inflammations Beware of pyroptosis that is accompanied by inflamation. It is mediated by caspase 1

Mechanism I. Four stages of apoptosis have been defined: i. Committment to death by extracellular or intracellular triggers/signals ii. Cell killing (execution) by activation of intracellular proteases (caspases) iii. Engulfment of cell corpse by other cells iv. Degradation of the cell corpse within the lysosomes of phagocytic cells

II. Stimuli for Apoptotic Cell Death in Mammals i. Growth factor deficiencies ii. Ionizing radiation/ viral infection iii. Free radical toxicity iv. Death receptor activation (such as Fas or CD95 triggering) v. Metabolic or cell cycle perturbation

Stimuli for Apoptotic Cell Death

Death Factors Definition: cytokines that activate an apoptosis program by binding to their specific receptor. Typical examples of death factors are: Fas ligand, TNF (tumor necrosis factor) and TRAIL (TNF-related apoptosis-inducing ligand). - Apoptosis can also be induced by cytotoxic T-lymphocytes using the enzyme granzyme. In some cases the apoptotic stimuli comprise extrinsic 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 such as cytotoxic T lymphocytes. The latter occurs when T-cells recognize damaged or virus infected cells and initiate apoptosis in order to prevent damaged cells from becoming neoplastic (cancerous) or virus-infected cells from spreading the infection. Apoptosis can also be induced by cytotoxic T-lymphocytes using the enzyme granzyme. The caspase cascade can be activated by: Granzyme B released by cytotoxic T lymphocytes which is known to activate caspase-3 and -7; death receptors (like FAS, TRAIL receptors and TNF receptor) which can activate caspase-8 and -10; and the apoptosome, regulated by cytochrome c and the Bcl-2 family, which activates caspase-9.

III. Activation of Caspase cascade i. Various stimuli described above eventually activate the executioner (caspase) cascade ii. At least 14 different caspases exist in human cells iii. Caspase cascades are apparently required for complete execution The caspase cascade can be activated by: Granzyme B released by cytotoxic T lymphocytes which is known to activate caspase-3 and -7; death receptors (like FAS, TRAIL receptors and TNF receptor) which can activate caspase-8 and -10; and the apoptosome, regulated by cytochrome c and the Bcl-2 family, which activates caspase-9.

Death Receptors In mammals, one mechanism by which initiation of cell death can occur is through interaction of death ligands, such as TNF-, FasL, or TNF-related apoptosis-inducing ligand (TRAIL). After interaction with their respective ligands, death receptors recruit adaptor proteins such as Fas-associated death domain protein (FADD) and TNF receptor–associated death domain protein (TRADD) to a plasma membrane complex called the death-inducing signaling complex. The extrinsic (death receptor-initiated) pathway of apoptosis, illustrated by the events following Fas engagement. FADD= Fas associated death domain.

Apoptotic body Cytoplasmic bleb Cell injury e.g., radiation , toxins, free radicals The Extrinsic pathway begins outside a cell, when conditions in the extracellular environment determine that a cell must die. The Intrinsic Apoptosis pathway begins when an injury occurs within the cell. Intrinsic stresses such as Oncogenes, direct DNA damage, Hypoxia, and survival factor deprivation Apoptotic body Cytoplasmic bleb

Genes: 1. antiapoptosis: Bcl-2, encodes a mitochondrial membrane protein/ 2. pro-apoptosis: p53. Two classes of IAP regulatory proteins are released from the mitochondria on receipt of an apoptotic signal. SMAC/DIABLO binds to the same pocket of XIAP as caspase 9. A second protein that is released from mitochondria, Omi/HtrA2, is a proapoptotic serine protease, which requires IAP binding and serine protease functions for activity. The intrinsic (mitochondrial) pathway of apoptosis. Death agonists cause changes in the inner mitochondrial membrane, resulting in the mitochondrial permeability transition (MPT) and release of cytochrome c and other pro-apoptotic proteins into the cytosol, which activate caspases. AIF= Apoptosis inhibitory factor; IAPs= Inhibitors of apoptosis proteins; Apaf-1= apoptosis protease activating factor

Stimuli that activate intrinsic pathway growth factor withdrawal, DNA damage, excessive growth signaling (oncogenes), viral infection and cell cycle abnormalities)

Initiator caspases include: 2, 8, 9, 10 Caspases are central initiators and executioners of apoptosis The term caspases is derived from cysteine-dependent aspartate-specific proteases: their catalytical activity depends on a critical cysteine-residue within a highly conserved active-site pentapeptide QACRG, and the caspases specifically cleave their substrates after Asp residues. Initiator caspases include: 2, 8, 9, 10 Execution caspases include: 3, 6, 7 necessary for apoptotic response, use cysteine residues to cleave peptide bonds following asprtate residues- cleave nuc envelope, cytoskeletal proteins, DNA repair enzymes and MAP kinases. Can inactivate IAPs and Bcl2

The caspase cascade can be activated by: Granzyme B released by cytotoxic T lymphocytes which is known to activate caspase-3 and -7; death receptors (like FAS, TRAIL receptors and TNF receptor) which can activate caspase-8 and -10; and the apoptosome, regulated by cytochrome c and the Bcl-2 family, which activates caspase-9. TNF-related apoptosis-inducing ligand (TRAIL).

Extrinsic apoptosis pathway formation of the Death Inducing Signaling Complex Death signals (Fas Ligand), TRAIL and TNF bind to death receptros to inititate apoptosis, death receptors are thransmembrane proteins that contain intracellular death domains, receptor-ligand binding stimulates fromation of DISC which contains the adaptor protein FADD and pro-initiator caspase 8...cleaved to become initiator caspase 8 Extrinsic apoptosis pathway (e.g. procaspase-8 is recruited by its DEDs to the death inducing signalling complex (DISC), a membrane receptor complex formed following to the ligation of a member of the tumor necrosis factor receptor (TNFR) family [Sartorius, 2001]. When bound to the DISC, several procaspase-8 molecules are in close proximity to each other and therefore are assumed to activate each other by autoproteolysis [Denault, 2002].

Intrinsic apoptosis pathway Intrinsic apoptosis pathway involve procaspase-9 which is activated downstream of mitochondrial proapoptotic events at the so called apoptosome, a cytosolic death signalling protein complex that is formed upon release of cytochrome c from the mitochondria [Salvesen, 2002]. In this case it is the dimerization of procaspase-9 molecules at the Apaf-1 scaffold that is responsible for caspase-9 activation [Denault, 2002]. Once the initiator caspases have been activated, they can proteolytically activate the effector procaspases-3, -6, and -7 which subsequently cleave a specific set of protein substrates, including procaspases themselves, resulting in the mediation and amplification of the death signal and eventually in the execution of cell death with all the morphological and biochemical features usually observed [Earnshaw, 1999].

Bcl-2 Bcl2 was the first apoptosis-related gene that was recognized to play a role in tumorigenesis, and indeed, Bcl-2 is overexpressed in a variety of cancers, contributing to cancer cell survival through direct inhibition of apoptosis. Desbribe how the balance between pro and anti-apoptotic Bcl-2 members determines whether the intrinsic pathway of apoptosis is activated All members contain BH3 domain. Anti-apoptosis: Bcl2 and BclX. Pro-Apoptosis: BH123 and BH3 only. BH123 normally kept inactive by Bcl2. Apoptosis signal, and BH3 starts working. BH3 binds to Bcl2, allowing BH123 to run wild and form oligomers in the mitochondrial membrane, leads to cyt c release...cyt c release allows formation of apoptosome and effector caspase cascade

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 membrane of the mitochondria. The gene was discovered as the translocated locus in a B-cell leukemia (hence the name). This translocation is also found in some B-cell lymphomas. 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.

Mitochondria as the regulator of cell death The activity of IAPs is inhibited by another protein called Second Mitochondrial-derived Activator of Caspases (SMAC), a natural antagonist of IAPs that acts as an endogenous regulator to enable apoptotic cell death. Cancer cells escape apoptosis when there is too little SMAC to overcome the elevated levels of IAPs.

Mimetics to induce cell death Venetoclax is an orally bioavailable, selective inhibitor of BCL2. It is a BH3 mimetic which acts similarly to the BH3-only proteins which antagonize BCL2 and its prosurvival family members

PARP, Poly-(ADP-ribose)-polymerase PARP, Poly-(ADP-ribose)-polymerase. The enzyme poly-(ADP-ribose) polymerase (PARP) is localized within the cell nucleus and catalyzes DNA-repair. During programmed cell death (apoptosis), PARP is enzymatically cleaved. Detection of the cleavage products is characteristic for apoptosis.

PARP, Poly-(ADP-ribose)-polymerase PARP, Poly-(ADP-ribose)-polymerase. The enzyme is involved in DNA repair and is inactivated during apoptosis by caspase-3 cleavage. The enzyme poly-(ADP-ribose) polymerase (PARP) is localized within the cell nucleus and catalyzes DNA-repair. During programmed cell death (apoptosis), PARP is enzymatically cleaved. Detection of the cleavage products is characteristic for apoptosis.

TUNEL: Terminal Uridine Nucleotide End Labeling X = conjugate i.e., biotin or digoxigenin. TdT (terminal deoxynucleotidyl transferase) catalyses the addition of nucleotides to the 3' terminus of a DNA molecule. Unlike most DNA polymerases it does not require a template. The preferred substrate of this enzyme is a 3'-overhang, but it can also add nucleotides to blunt or recessed 3' ends. TUNEL: Terminal Uridine Nucleotide End Labeling