CELL DEATH & APOPTOSIS

CELL DEATH & APOPTOSIS : 

CELL DEATH & APOPTOSIS BY :YASHWANTH B.M 1 st M.PHARM 1

DEFINATION :: 

DEFINATION : Cell death is a state of irreversible injury. It may occur in the living body as a local or focal change (i.e., autolysis, necrosis and apoptosis) and the changes that follow it (i.e., gangrene and pathologic calcification) or result in end of the life (somatic death). These pathologic processes involved in the cell death are as follows . 2

CELL DEATH: 

CELL DEATH • Cells die by one of two mechanisms – necrosis or apoptosis. • Two physiologically different processes – Necrosis & autolysis – death by injury – Apoptosis – death by suicide • Apoptosis and necrosis have different characteristics. 3

AUTOLYSIS: 

AUTOLYSIS Autolysis (self digestion) is disintegration of the cell by its own hydrolytic enzymes liberated from lysosomes . It occurs in the living body in places surrounded by inflammatory reaction or it may occur as post-mortem change in which there is complete absence of surrounding inflammatory response. Tissues rich in hydrolytic enzymes such as in pancreas and gastric mucosa autolysis is rapid . Morphologically : the cell is swollen, membrane is intact & cytoplasm is homogeneous & eosinophilic with cell debris or loss of cellular details. 4

NECROSIS: 

NECROSIS It is defined as focal death along with protein denaturation & degradation of tissue by hydrolytic enzymes liberated by cells. It is accompanied by inflammatory reaction. Necrosis is caused by various agents such as hypoxia, chemical and physical agents, microbial agents and immunological injury Two essential changes bring about irreversible cell injury in necrosis-cell digestion by lytic enzymes and denaturation of proteins 5

TYPES OF NECROSIS :: 

TYPES OF NECROSIS : a) Coagulative necrosis: This type of necrosis is caused by irreversible focal injury, mostly from sudden cessation of blood flow ( ischaemia ) and less often from bacterial and chemical agents. Generally heart , kidney and spleen are affected. 6

(b)Liquefaction (Colliquative) necrosis: : 

(b)Liquefaction ( Colliquative ) necrosis: 7 This type of necrosis occurs commonly due to ischaemic injury and bacterial or fungal infections. . It occurs due to degradation of tissues by the action of powerful hydrolytic enzymes eg : infarct brain and abscess cavity.

Caseous necrosis: 

Caseous necrosis It is found in the centre of foci of tuberculous infections .it combines features of both coagulative and liquefactive necrosis . 8

Fat necrosis: 

Fat necrosis It is a special form of cell death occurring at two anatomically different locations but morphologically similar lesions. These are acute pancreatic necrosis and traumatic fat necrosis (commonly in breast). 9

Fibrinoid necrosis: 

Fibrinoid necrosis It is characterised by deposition of fibrin like material which has the staining properties of fibrin. It is encountered in various examples of immunologic tissue injury ( Eg in immune complex vasculitis , autoimmune disease, Arthurs reaction etc..,) arterioles in hypertension, peptic ulcer etc. 10

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11 APOPTOSIS

CONTENTS :: 

CONTENTS : History. Definition. Significance of apoptosis. Apoptosis Vs Necrosis. Morphological features. Molecular Mechanism . 12

HISTORY OF APOPTOSIS :: 

HISTORY OF APOPTOSIS : 1858 - Virchow - Cell vanishes and can no longer be seen in its orginal form - Necrobiosis 1885 – Walther Flemming – Regression of epithilium cells - Chromatolysis 1914 – Ludwig Graper - First review on Chromatolysis 1951 – Glucksmann – Chromatosysis in embryo Development level 1972 – Kerr, Wyllie, Curie – Apoptosis 13

APOPTOSIS: 

APOPTOSIS Apoptois or programmed cell death is a form of internally programmed coordinated cell death which is induced by a tightly regulated intracellular program in which cells destined to die activate intracellular enzymes that degrade the cells' own nuclear DNA and nuclear and cytoplasmic proteins. Apoptosis in Greek means “falling off” or “dropping off”. 14

Significance of apoptosis:: 

Significance of apoptosis: It serves to eliminate unwanted, worn out potentially harmful cells which have outlived their usefulness. Serves to eliminate cells with irreparable faulty DNA produced as a result of mutation etc, thereby preventing proliferation of defective cells. Helps in cessation of immune response. During development many cells are produced in excess which eventually undergo programmed cell death and thereby contribute to sculpturing many organs and tissues. 15

Apoptosis vs. Necrosis :: 

Apoptosis vs. Necrosis : Apoptosis Chromatin condensation Cell shrinkage & membrane blebbing . Preservation of organelles and cell membranes Rapid engulfment by Macrophages preventing inflammation. Necrosis Nuclear swelling Cell swelling Disruption of organelles Rupture of cell and release of cellular contents Inflammatory response 16

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Morphological features:: 

Morphological features: Proteolysis of cytoskelital proteins( actin & lamnin ) &Cross linking of proteins. Fragmentation of nucleus& lysis of nuclear chromatin by nuclease. Formation of membrane blebs & cell shrinkage formation of cell fragments called apoptotic bodies. Appearance of phosphatidylserine & thrombospondin on outer cell membrane serving as marker proteins for phagocytes ( macrophages & monocytes ). 18

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Image below shows a trophoblast cell undergoing apoptosis.: 

Image below shows a trophoblast cell undergoing apoptosis . Cell begins to shrink following the cleavage of lamins and actin filaments of the cytoskeleton. (A) The breakdown of chromatin in the nucleus leading to nuclear condensation & 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. (D) Small vesicles called apoptotic bodies are also sometimes observed. 20

Biochemical features of apoptosis :: 

Biochemical features of apoptosis : Protein cleavage : activation of pro- caspase to caspase which cleave actin & lamnin filaments of cytoskeliton . Caspase mediated activation of DNAase causing nuclear pyknosis (condensation) & karyorrhexis (fragmentation). DNA breakdown : Ca-Mg dependant exonucleases breakdown DNA into fragments of 50-300 kilobasepieces with further intranucleosomal cleavage of DNA into oligonucleosomes in multiples of 180-200 basepairs . Phagocytic recognition: expression of phosphatidylserene & thrombospondin on outer cell membrane along with opsonization lead to their early recoghnition & elimination by phagocytes without any inflammatory response. 21

Identifying apoptotic cells :: 

Identifying apoptotic cells : Staining of condensed chromatin : by haematoxylin , feulgen , acridene orange. Flow cytometry : to visualize rapid cell shrinkage . Gel electrophoresis : to detect DNA fragmentation(DNA laddering test) Annexin V dye : marker for phosphatydyl serene on cell membrane of apoptotic bodeis . 22

Measurement of apoptosis: 

Measurement of apoptosis Phase contrast microscopy Determination of ADP / ATP ratio Tunnel assay( TdT mediated dUTP nick end labeling assay ) FITC ( Flourescein isothiocynate ) DNA laddering test 23

Apoptosis in Physiologic Situations :: 

Apoptosis in Physiologic Situations : Embryogenesis Metamorphosis of Tadpole into frog 24

Apoptosis in Physiologic Situations :: 

Apoptosis in Physiologic Situations : Hormone-dependent involution in the adult ex: endometrial shedding , regression of lactating breast, involution of thymus in adults, involution of appendix. Cell deletion in proliferating cell population : ex intestinal crypt epithelial cells, to maintain a steady cell count. Elimination of self reactive lymphocytes: Elimination of CD4 ( HelperTcells ) &CD-8( KillerT cells) after immune response. 25

Apoptosis in Pathologic Conditions: 

Apoptosis in Pathologic Conditions Cell death produced by a variety of injurious stimuli ex: chemotherapeutic agents , radiation, physical & biological agents (viral diseases). Atrophy in parenchymal organs ex :duct obstruction of pancreas, parotid gland, kidney, prostatic atrophy ( orchidectomy ). Degenerative diseases of CNS ex: aizheimers’s disease, parkinsonism, etc… Cell death by lymphocytes during immune response. Reduced CD-4 &CD-8 cell count in AIDS. 26

Initiators of Apoptosis: : 

Initiators of Apoptosis: Absence of survival stimuli : (a)growth factor(neurons,blood cell etc )& other hormones ex: sex hormones during menstruation. (b) cytokines,antigens , ex: CD4 &CD8 cells. Genotoxic stress: hypoxia, heat, radiation etc… leading to DNA damage & activation of P53 gene causing apoptosis (intrinsic pathway). Activation of TNF death rec : ( extrinsic pathway). 27

Initiators of Apoptosis: : 

Initiators of Apoptosis: CD-8 Tcell mediated apoptosis: release TNF- α responsible for extrinsic pathway. & 28 CTL,s release perforin & CTL granule containing granzyme -B Proteolysis at aspartate residues Caspace cascade Pro- caspase to caspase granzyme -B granzyme -B

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MOLECULAR MECHANISM:: 

MOLECULAR MECHANISM: INITIATOIN : (A) intrinsic pathway (B) extrinsic pathway EXECUTION PHASE: PHAGOCYTOSIS : 30

(A) Intrinsic pathway: 

(A) Intrinsic pathway Intrinsic pathway a balance b/w pro & anti-apoptotic molecules that regulate mitochondrial permeability. Regulators of apoptosis : Bcl-2 family consisting of 20 types of protein of which Bcl-2 & Bcl -x are anti-apoptotic molecules while Bax , Bak , Bim , Bad, are pro-apoptotic . Initiating signals Genotoxic stress or Absence of survival stimuli inactivates anti-apoptotic molecules while synthesis of P53 (guardian of genome) occours , ATM is activated which signals for Dephosphorylation & activation of Bax , Bak , Bim , Bad. 31

Intrinsic pathway: 

Intrinsic pathway Bax , Bak , Bim & Bad Increase mitochondrial membrane permiablity & leakage of Ca2+,H+, cytochrome -c. cytochrome -c & Apaf-1 complex activates pro capase-9 to capase-9 ( apoptosome formation). capase-9 activates other procaspaces & initiates caspase cascade. 32

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The Apoptosome : 

The Apoptosome Granzyme B delivered into cells by cytotoxic T lymphocytes and is able to directly activate caspases 3, 7, 8 and 10. The mitochondria release of cytochrome C from mitochondria can lead to the activation of caspase 9, and then of caspase 3. This effect is mediated through the formation of an apoptosome , a multi-protein complex consisting of cytochrome C, Apaf-1, pro- caspase 9 and ATP. 34

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(B) Extrinsic pathway: 

(B) Extrinsic pathway Death Receptors : Death receptors are cell surface receptors that transmit apoptotic signals initiated by specific ligands such as Fas ligand , TNF alpha and TRAI-L. They can activate a caspase cascade within seconds of ligand binding. Induction of apoptosis via this mechanism is therefore very rapid. 36

Apoptotic signalling from the death receptors: : 

Apoptotic signalling from the death receptors: Binding of the ligand to its receptor can lead to a the generation of ceramide , produced by hydrolysisof sphingomyelin . This ceramide release is thought to promote clustering of the death receptors. The large scale receptor clustering is important because it helps amplify the apoptotic signalling . In the absence of receptor clustering some cells, such as lymphocytes, are still able to trigger apoptosis. 37

Apoptotic signalling from the death receptors:: 

Apoptotic signalling from the death receptors : Following ligand binding a conformational change in the intracellular domains called "death domain“ allows the recruitment of various apoptotic proteins to the receptor. This protein complex is often called the DISC, or Death Inducing Signalling Complex. The final step in this process is the recruitment of one of the caspases , typically pro- caspase 8, to the DISC. This results in activation of caspase 8 and the inititation of apoptosis. 38

TNF receptor signalling : : 

TNF receptor signalling : TNF is produced by T-cells and activated macrophages in response to infection. Binding of TNF alpha to TNFR1 results in receptor trimerisation and clustering of intracellular death domains. This allows binding of an intracellular adapter molecule called TRADD (TNFR-associated death domain) via interactions between death domains. TRADD has the ability to recruit a number of different proteins to the activated receptor. TRADD can also associate with FADD, which leads to the induction of apoptosis via the recruitment and cleavage of pro- caspase 8 to caspase 8. Capase cascade follows. 39

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Signaling by Fas (CD95) : 

Signaling by Fas (CD95) The ligand for Fas ( FasL or CD95L) activates apoptosis in a similar way to the TNF receptor. Binding of the ligand promotes receptor clustering. The adapter protein FADD can be recruited directly to the death domain on the Fas receptor, without requiring the prior recruitment of TRADD. Caspase-8 activation & Caspase cascade. In addition the Fas receptor is generally though to only activate apoptosis . 41

EXECUTION PHASE:: 

EXECUTION PHASE: Caspace-8&9 are initiator caspases which through caspase cascade inactivate various proteins while activating caspace-3&6 serving a exicutioners . Caspases and chromatin breakdown Caspase activated Ca-Mg dependant exonucleases breakdown DNA into fragments of 50-300 kilobasepieces with further intranucleosomal cleavage of DNA by activating CAD, or caspase activated DNase . Normally CAD exists as an inactive complex with ICAD (inhibitor of CAD). During apoptosis, ICAD is cleaved by caspase 3, to release CAD. Rapid fragmentation of the nuclear DNA follows. 42

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44 Inactivation of enzymes involved in DNA repair. The enzyme poly (ADP-ribose) polymerase, or PARP is involved in repair of DNA damage. The ability of PARP to repair DNA damage is prevented following cleavage of PARP by caspase-3. 2 ) Breakdown of structural nuclear proteins. Actin & Lamins are that maintain the shape of the cell. Degradation of nuclear matrix proteins & cytoskelital proteins by caspase 6 results in the chromatin condensation and nuclear fragmentation commonly observed in apoptotic cells. Oligonucleosomes ( nucleosomal units) exist in multiples of 180-200 basepairs .

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PHAGOCYTOSIS :: 

PHAGOCYTOSIS : : The dead apoptotic cells and their fragments possess cell surface receptors phosphatidylserene & thrombospondin on outer cell membrane which along with opsonization facilitates their identification by adjacent phagocytes& lead to their early recognition & elimination by phagocytes without any inflammatory response. 46

REFERENCES:: 

REFERENCES: 1) PRINCIPLES OF ANATOMY AND PHYSIOLOGY - TORTORA & GRABOWSKI 2) TEXTBOOK OF PATHOLOGY – HARSH MOHAN 3) ROBBINS COTRAN PATHOLOGIC BASIS OF DISEASES 4) INTERNET SOURCE – WIKIPEDIA 47

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48 THE END