1. ASSIGNMENT ON APOPTOSIS Rajiv Gandhi Proudyogiki Vishwavidyalaya
Submitted to
Rajiv Gandhi Proudyogiki Vishwavidyalaya
Supervised by : Submitted by :
Dr. Neelam Balekar Simarpeet Kaur Bhatia
M.Pharm, PhD M.Pharm (1st SEM)
Professor & Vice Principal
COP, IPS ACADEMY, INDORE

2. INTRODUCTION
It is a functional death and it is a good mechanism to eliminate wasted, useless, unwanted, or crippled cells.
Process of programmed cell death that may occur in multicellular organisms
Leads to characteristic changes like cell shrinkage, nuclear fragmentation, chromatin condensation & chromosomal DNA fragmentation
Between 50 & 70 billion cells die each day due to apoptosis in the average human adult.

3. HISTORY
German scientist Carl Vogt was first to describe the principle of apoptosis in 1842
In 1885, anatomist Walther Flemming delivered a more precise description of the process of programmed cell death.
Kerr received the Paul Ehrlich & Ludwig Darmastedter Prize on March 14, 2000, for his description of apoptosis.
The 2002 Nobel Prize in medicine was awarded to Sydney Brenner, Horvitz and John E. Sulston for their work regarding apoptosis.

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
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
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. NECROSIS Vs APOPTOSIS
Wilde, 199

7. STAGES OF APOPTOSIS
Induction of apoptosis related genes, signal transduction

8. APOPTOSIS: Morphology
organelle
reduction
membrane blebbing & changes
cell
shrinkage
mitochondrial leakage
nuclear fragmentation
chromatin condensation

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

10. Blebbing & Apoptotic bodies
The control retained over the cell membrane & cytoskeleton allows intact pieces of the cell to separate for recognition & phagocytosis by MFs
Apoptotic body MF

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

13. MAJOR PLAYERS IN APOPTOSIS
Caspases
Adaptor proteins
TNF & TNFR family
Bcl2 family

14. Ligand-induced cell death
Ligand Receptor FasL Fas (CD95) TNF TNF-R TRAIL DR4 (Trail-R)

15. Ligand-induced cell death
“The death receptors”
Ligand-induced trimerization
FasL
Trail
TNF
Death Domains
Death Effectors
Induced proximity of Caspase 8
Activation of Caspase 8

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

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

18. The mitochondrial pathway
Growth factor
receptors
DNA
damage
Fas
Casp8
PI3K
p53
Akt
Bid
casp3
BAD
Bid
Bax
IAPs
casp9
Bid
Bcl2
Apaf1
ATP
casp3
Bax
Cyt.C
Smac/
DIABLO
H2O2
AIF

19. REGULATION OF APOPTOSIS
Stimuli apoptosis selection of targets
Apoptosis by conflicting signals that scramble the
normal status of cell
Apoptotic stimulicytokines, death factors (FasL)
DNA breaks  p53 is activated arrest cell cycle or
activate self destruction )

20. MEASUREMENT OF APOPTOSIS:
TECHNIQUES BASED ON MORPHOLOGICAL CHANGES
Light microscopy
Electron microscopy
TECHNIQUES BASED ON DNA FRAGMENTATION
Measurement of endonuclease activity

21. MEASUREMENT OF APOPTOSIS:
TECHNIQUES BASED ON MEMBRANE ALTERATIONS
Measurement of dye exclusion
TECHNIQUES BASED ON CYTOPLASMIC CHANGES
Changes in intracellular enzyme activity
Measurement of calcium influx

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. APOPTOSIS: important in embryogenesis
Morphogenesis (eliminates excess cells):
Selection (eliminates non-functional cells):

24. APOPTOSIS: important in embryogenesis
Immunity (eliminates dangerous cells):
Self antigen
recognizing cell
Organ size (eliminates excess cells):

25. APOPTOSIS: important in adults
Tissue remodeling (eliminates cells no longer needed):
Apoptosis
Late pregnancy, lactation
Involution
(non-pregnant, non-lactating)
Virgin mammary gland
- Testosterone
Apoptosis
Prostate gland

26. APOPTOSIS: important in adults
Tissue remodeling (eliminates cells no longer needed):
Apoptosis
Resting lymphocytes
+ antigen (e.g. infection)
- antigen (e.g. recovery)
Steroid immunosuppressant: kill
lymphocytes by apoptosis
Lymphocytes poised to die by apoptosis

27. APOPTOSIS: important in adults
Maintains organ size and function: X
Apoptosis
+ cell division
Cells lost by apoptosis are replaced by cell division

28. APOPTOSIS: Role in Disease
TOO MUCH: Tissue atrophy
Neurodegeneration,
Thin skin
etc
TOO LITTLE: Hyperplasia
Cancer
Atherosclerosis
etc

29. APOPTOSIS: Role in Disease
Neurodegneration:
Neurons are post-mitotic (cannot replace themselves;
neuronal stem cell replacement is inefficient)
Neuronal death caused by loss of proper connections,
loss of proper growth factors (e.g. NGF), and/or
damage (especially oxidative damage)
Neuronal dysfunction or damage results in loss of synapses
or loss of cell bodies
(synaptosis is reversible; apopsosis is irreversible)
Also plays a major role in Pakinson’s disease, Alzheimer’s disease & Huntington’s disease

30. APOPTOSIS: Role in Disease
Cancer
Apoptosis eliminates damaged cells
(damage => mutations => cancer
Tumor suppressor p53 controls senescence
and apoptosis responses to damage
Most cancer cells are defective in apoptotic response
(damaged, mutant cells survive)
High levels of anti-apoptotic proteins or
Low levels of pro-apoptotic proteins causes cancer

31. APOPTOSIS: Role in Disease
Aging
Aging --> too much and too little apoptosis
(evidence for both)
If it is too much (accumulated oxidative damage)
---> tissue degeneration
If it is too little (defective sensors, signals)
---> dysfunctional cells accumulate

32. 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

33. 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.

34. Thank you