This document summarizes programmed cell death or apoptosis. It discusses the distinct modalities of programmed cell death including apoptosis, autophagy and necroptosis. It covers the role of programmed cell death in disease, the mechanisms and molecular regulators involved in different types of cell death, methods to detect programmed cell death, and the therapeutic potential of modulating programmed cell death pathways.

1. Programmed Cell Death
Presented by-
Shipra Kartik
Supervised by-
Dr. Rishi Pal,
Associate professor,
Dept. of Pharmacology,
King George’s Medical University, Lucknow.

2. Content
 Introduction
 Distinct modalities of programmed cell death (PCD)
 Role of PCD in disease
 Mechanism of PCD
 Detection of PCD
 Therapeutic potential of PCD
 Conclusion

3. Introduction
 Definition- Programmed cell death is a genetically
regulated process of cell suicide that is central to the
development, homeostasis and integrity of multicellular
organisms.
 Etiology of cell death-
 Cell death by injury
-Mechanical damage
-Exposure to toxic chemicals
 Cell death by suicide
-Internal signals
-External signals

4. Why do cell needs to die?

5. History of Cell death research
• 1800s Numerous observation of cell death
• 1908 Mechnikov wins Nobel prize (Phagocytosis)
• 1930-40 Studies of metamorphosis
• 1964-66 de Duve coined autophagy and Necrosis & PCD described
• 1971 Term apoptosis coined
• 1977 Cell death genes in C. elegans
• 1980-82 DNA ladder observed & ced-3 identified
• 1989-91 Apoptosis genes identified, including bcl-2, fas/apo1 & p53, ced-3
sequenced
• 1995–1996 The signalling pathway for FASL-induced apoptosis was elucidated
• 1997 The Oshumi group clone yeast ATG-1and 30 other ATG gene.
 2002 The Nobel Prize to Sydney Brenner, H.Robert Horvitz and John E.
Sulston for programmed cell death or Apoptosis.
 2005 A form of necrosis that could be inhibited by the chemical
necrostatin 1 was identified and termed ‘necroptosis’
• 2012–2014 MLKL was shown to execute necroptosis downstream of RIPK1
and RIPK3
• 2016 The Nobel prize to Yoshinori Ohsumi for Autophagy.

6. Distinct modalities of Programmed cell death
Cell Death Differ. 2009, 16(1): 3–11

7. Galluzzi et al.
Cell Death & Differentiation (2012)

8. Role of PCD in diseases
• Important in normal physiology / development
Development: Immune systems maturation, Morphogenesis,
Neural development
Adult: Immune privilege, DNA Damage and wound repair.
• Excessive Cell death
Neurodegenerative diseases
Aging
• Deficient Cell death
Cancer
Autoimmunity

9. Apoptosis and diseases

10. Autophagy and diseases

11. Necroptosis and diseases

12. Mechanism of PCD
Apoptosis
• Apoptosis (or type I programmed cell death) is a genetic pathway for rapid
and efficient killing of unnecessary or damaged cells that was initially
described by Vogt (1842).
• Inducers of Apoptosis-
Apoptosis occurs during normal cell turnover, tissue homeostasis,
embryogenesis, induction and maintenance of immune tolerance.
• Types of apoptotic cell death-
 Intrinsic (mitochondrial mediated) pathway.
 Extrinsic (death receptor mediated) pathway.
 Perforin/ Granzyme pathway.
www.chembio.uoguelph.ca

13. Apoptosis in C.elegans
•The life cycle of C. elegans from egg to sexual maturity (and new eggs)
is about 3 days. Ced-1, -3, -4, and -9 (Cell death determining) proteins in
C.elegans are closely related to mammalian apoptosis- regulating genes
•Caenorhabditis elegans consist 1090 somatic cells are generated in the
formation of the adult worm, of which 131 of these cells undergo
apoptosis.
www.chembio.uoguelph.ca

14. Morphology of Apoptosis

15. Methods Enzymol. 2008;442:157-81

16. Caspase-dependent and -independent ‘intrinsic apoptosis’
Galluzzi et al.
Cell Death & Differentiation (2012) 19, 107–

17. Galluzzi et al.
Cell Death & Differentiation (2012) 19, 107–120
Extrinsic Apoptotic Pathway

18. The target sequence for Ced-3 and caspases
(Cys catalytic Asp targeting proteases)
consists of a tetrapeptide with C-terminal Asp (D).
Molecular regulators of apoptosis
Role of caspases

19. Bcl-2 family: Pro-Life and Pro-Death factions
www.chembio.uoguelph.ca
Caspase-9
Effector Caspase

20. Genetic control of Apoptosis
S No. Apoptotic gene Cellular location of protein products Effect on apoptosis
1. Bcl-2 Mitochondrial membrane Block
2. myc Nuclear envelop
Endoplasmic reticulum
Stimulate
3. P53 Nucleus Wild type
Stimulates
4. P53* Nucleus Mutant block
5. APO-1/ FAS Cell membrane Stimulates

21. Galluzzi et al.
Cell Death & Differentiation (2012) 19, 107–120
The ability of cells to digest their own cellular components was first observed
by Christian de Duve, who subsequently introduced the term ‘autophagy’ to
describe the process.
Autophagy

22. Different types of autophagic
pathway
Macroautophagy
Microautophagy
Chaperone-mediated autophagy
Molecular regulators of Autophagy
 Induction (e.g. Atg1 and ULK1)
Membrane expansion (Atg 8/ LC3, Atg 12)
 Autophagosome formation (Atg 5,8,9,12,16, Beclin-1)
 Degradation acid hydrolase degrades the cargos (e.g. Atg 9 and cathepsin
B, D,L)

23. Galluzzi et al.
Cell Death & Differentiation (2012) 19, 107–120
Regulated Necrosis: Necroptosis
Necroptosis is referred to as cell death initiated by TNF receptors following
chemical suppression of caspases. Peter Vandenabeele and Junying Yuan were
the first authors to describe the morphological and biochemical features of
necroptosis.

24. Molecular regulators of Necroptosis
 RIPK(receptor-interacting serine/threonine protein kinase)
 RIPK1*
 RIPK3*
• Death execution factors
 MLKL (mixed-lineage kinase domain-like)
 Ion channels
 Lipid peroxidation and lysosomal and plasma- membrane
permeabilization.
• Death regulatory factors

25. Nature Immunology 4, 416 - 423 (2003)
Role of Mitochondria in PCD

27. Detection of PCD
Guidelines for the use and interpretation of PCD

28. Biochemical aspects of distinct cell modalities
S No. Cell death mode Biochemical features
1. Apoptosis  Activation of proapoptotic Bcl-2 family proteins (e.g., Bax, Bak,
Bid)
 Activation of caspases
 ∆Ψm dissipation
 Oligonucleosomal DNA fragmentation
 Plasma membrane rupture
 PS exposure
 ROS overgeneration
 ssDNA accumulation
2. Autophagy
 Beclin-1 dissociation from Bcl-2/XL
 Dependency on atg gene products
 LC3-I to LC3-II conversion
3. Necroptosis
 Activation of Cathepsins and Calpains
 HMGB-1 release
 Plasma membrane rupture
 RIP1 ubiquitination
 ROS overgeneration
 Specific PARP1 cleavage pattern

29. Detection of apoptotic changes in DNA
• Nucleic acid staining – nuclear morphology
• Detection of nuclear DNA fragmentation
• TUNEL staining (terminal deoxynucleotidyl transferase–
mediated dUTP nick end-labeling)
• Single-cell electrophoresis (Comet assay)
MolecularProbes,Inc.

30. Detection of changes in cell membrane integrity
• Membrane permeability
• Phospholipid symmetry (Annexin V staining)
MolecularProbes,Inc.

31. • Detection of apoptotic changes in mitochondria:
• Caspase Protease Assays (individual caspases):
Caspase activation is detected by cell-permeant
fluorogenic substrates.
MolecularProbes,Inc.
Morphology MPT

32. Detection of anti-apoptosis proteins.
Detecting changes in gene expression for pro- and anti-apoptosis genes

33. Therapeutic potential of PCD
 Interfere signal transduction, signal molecules, receptors,
2nd messengers, etc
 Regulate apoptosis-related enzymes and genes
 Prevent decrease in mitochondria trans-membrane potential
 Others

35. Conclusion
 “Programmed cell death” can be activated by moderate
stress which has damaged the cell beyond its ability to
recover fully or by viral infection.
 This has the desirable effect of removing damaged or
infected cells.
 Selective manipulation of cell death pathways may be an
important approach for treating cancer, neurodegenerative
and autoimmune diseases in the future.