1. APOPTOSIS: An overview Sanjeev Sharma*, Aarti Bhardwaj $, Shalini Jain # and Hariom Yadav # *Animal Genetics and Breeding Division, # Animal Biochemistry Division, National Dairy Research Institute, Karnal-132001, Haryana, India $ College of Applied Education and Health Sciences, Meerut, U.P.

2. INTRODUCTION Cell death by injury -Mechanical damage -Exposure to toxic chemicals Cell death by suicide -Internal signals -External signals

3. Conted….. Apoptosis or programmed cell death, is carefully coordinated collapse of cell, protein degradation, DNA fragmentation followed by rapid engulfment of corpses by neighbouring cells. (Tommi, 2002) Essential part of life for every multicellular organism from worms to humans. (Faddy et al.,1992) Apoptosis plays a major role from embryonic development to senescence.

4. Why should a cell commit suicide? Apoptosis is needed for proper development Examples: –The resorption of the tadpole tail –The formation of the fingers and toes of the fetus –The sloughing off of the inner lining of the uterus –The formation of the proper connections between neurons in the brain Apoptosis is needed to destroy cells Examples: –Cells infected with viruses –Cells of the immune system –Cells with DNA damage –Cancer cells

5. What makes a cell decide to commit suicide? Withdrawal of positive signals examples : –growth factors for neurons –Interleukin-2 (IL-2) Receipt of negative signals examples : –increased levels of oxidants within the cell –damage to DNA by oxidants –death activators : Tumor necrosis factor alpha (TNF-  ) Lymphotoxin (TNF-β) Fas ligand (FasL )

6. History of cell death / apoptosis research 1800sNumerous observation of cell death 1908Mechnikov wins Nobel prize (phagocytosis) 1930-40Studies of metamorphosis 1948-49Cell death in chick limb & exploration of NGF 1955Beginning of studies of lysomes 1964-66Necrosis & PCD described 1971Term apoptosis coined 1977Cell death genes in C. elegans 1980-82DNA ladder observed & ced-3 identified 1989-91Apoptosis genes identified, including bcl-2, fas/apo1 & p53, ced-3 sequenced (Richerd et.al., 2001)

7. Necrosis vs. Apoptosis Cellular condensation Membranes remain intact Requires ATP Cell is phagocytosed, no tissue reaction Ladder-like DNA fragmentation In vivo, individual cells appear affected Cellular swelling Membranes are broken ATP is depleted Cell lyses, eliciting an inflammatory reaction DNA fragmentation is random, or smeared In vivo, whole areas of the tissue are affected NecrosisApoptosis

8. NECROSIS Vs APOPTOSIS Wilde, 1999

9. STAGES OF APOPTOSIS Sherman et al., 1997 Induction of apoptosis related genes, signal transduction

10. membrane blebbing & changes mitochondrial leakage organelle reduction cell shrinkage nuclear fragmentation chromatin condensation APOPTOSIS: Morphology Hacker., 2000

11. membrane blebbing & changes mitochondrial leakage organelle reduction cell shrinkage nuclear fragmentation chromatin condensation APOPTOSIS: Morphological events

12. Bleb Blebbing & Apoptotic bodies The control retained over the cell membrane & cytoskeleton allows intact pieces of the cell to separate for recognition & phagocytosis by M  s Apoptotic body MM MM

13. Caenorhabditis elegans 1090 cells131 cellsapoptosis ced-1 ced-2 ced-5 ced-6 ced-7 ced-10 ced-3 ced-4 ced-9egl-1 ces-1ces-2 nuc-1 execution decision to die engulfmentdegradation

14. Apoptosis: Pathways Death Ligands Effector Caspase 3 Death Receptors Initiator Caspase 8 PCD DNA damage & p53 Mitochondria/ Cytochrome C Initiator Caspase 9 “Extrinsic Pathway” “Intrinsic Pathway”

15. MAJOR PLAYERS IN APOPTOSIS Caspases Adaptor proteins TNF & TNFR family Bcl-2 family

16. Ligand-induced cell death LigandReceptor FasLFas (CD95) TNFTNF-R TRAILDR4 (Trail-R)

17. Ligand-induced cell death “The death receptors” Ligand-induced trimerization Death DomainsDeath EffectorsInduced proximity of Caspase 8 Activation of Caspase 8 FasL Trail TNF

18. p53 Apoptosis events Initiator caspases 6, 8, 9,12 Activators of initiator enzymes Apoptotic signals Execution caspases 2, 3, 7 APOPTOSIS: Signaling & Control pathways I Externally driven Internally driven Cytochrome C Externally driven Activation mitochondrion

19. p53 External Internal Apoptosis events Initiator caspases 6, 8, 9,12 Activators of initiator enzymes Apoptotic signals Execution caspases 2, 3, 7 Inhibitors of apoptosis APOPTOSIS: Signaling & Control pathways II Inhibitors Externally driven Internally driven Cytochrome C Externally driven Survival factors Bcl2 Inhibition

20. H2O2H2O2 Growth factor receptors casp9 Bcl2 PI3K Akt BAD Apaf1 Cyt.C ATP The mitochondrial pathway casp3 IAPs Smac/ DIABLO AIF Bax p53 Fas Casp8 Bid DNA damage Pollack etal., 2001

21. REGULATION OF APOPTOSIS Stimuli  apoptosis  selection of targets (Rich et al., 2000) Apoptosis by conflicting signals that scramble the normal status of cell (Canlon & Raff, 1999) Apoptotic stimuli  cytokines, death factors (FasL) (Tabibzadeh et al., 1999) DNA breaks  p53 is activated  arrest cell cycle or activate self destruction (Blaint & Vousden, 2001)

22. Importance of Apoptosis Important in normal physiology / development –Development: Immune systems maturation, Morphogenesis, Neural development –Adult: Immune privilege, DNA Damage and wound repair. Excess apoptosis –Neurodegenerative diseases Deficient apoptosis –Cancer –Autoimmunity

23. FUTURE PERSPECTIVES The biological roles of newly identified death receptors and ligands need to be studied Need to know whether defects in these ligands and receptors contribute to disease

24. CONCLUSION an important process of cell death can be initiated extrinsically through death ligands (e.g. TRAIL, FasL) activating initiator caspase 8 through induced proximity. can be initiated intrinsically through DNA damage (via cytochrome c) activating initiator caspase 9 through oligomerization. Initiator caspases 8 and 9 cleave and activate effector caspase 3, which leads to cell death.

27. DNA DAMAGE p53

28. The bcl-2 family BH4BH3BH1BH2TM NC Receptor domain phosphorylation Raf-1 calcineurin Pore formation Membrane anchor Ligand domain Group I Group II Group III Bcl-2 bax Bad bid bik Back

29. P53 & Apoptosis p53 first arrests cell growth between G1  S This allows for DNA repair during delay If the damage is too extensive then p53 induces gene activation leading to apoptosis (programmed cell death) BACK

30. 3 mechanisms of caspase activation a. Proteolytic cleavage e.g. pro-caspase 3 b. Induced proximity, e.g. pro-caspase 8 c. Oligomerization, e.g. cyt c, Apaf-1 & caspase 9 Back

31. Cytolytic lymphocyte/CTL (& natural killer lymphocyte) presents Fas ligand/CD178 on its surface to tell the infected cell to die Apoptosis events Initiator caspases Apoptotic signals Execution caspases Externally driven Cytochrome c Fas ligand Apoptosis signal to kill infected cells CTL Virally infected cell Fas/ CD95 is the ‘death receptor’ The immunological synapse holds the cells much tighter together than shown here