its about pathological basics in apoptosis in health and disease.... pg level

1. APOPTOSIS IN HEALTH
& DISEASES
MODERATOR : PROF. PARAMESH
PRESENTER : DR. MEENU . E.V.

2. INTRODUCTION

3. Apoptosis - Definition
• A pathway of cell death induced by a tightly
regulated suicidal program, in which the
cells destined to die activate enzymes that
degrade cells own nuclear DNA and nuclear,
cytoplasmic proteins.
sclero dinesh

4. Kerr Wyllie and Currie paper, British Journal of Cancer, 1972 Aug;26(4):239-57
"We are most grateful to Professor James
Cormack of the Department of Greek,
University of Aberdeen, for suggesting this
term. The word "apoptosis" (ἁπόπτωσισ) is
used in Greek to describe the "dropping off"
or "falling off" of petals from flowers, or
leaves from trees”.
mev

5. Historical aspects
• German scientist Carl Vogt - Principle of apoptosis (1842).
Mev

6. • Walther Flemming – Process of programmed
cell death (1845).
mev

7. • John Foxton Ross Kerr – Distinguish
apoptosis from traumatic cell death (1962).
mev

8. Nobel prize in 2002 – Sydney Brenner , Horvitz, John
Buston.
‘identified gene that control apoptosis’
Study done in “Caenorhabditis Elegans”

9. Significance of apoptosis
• During development many cells are produced in excess which
eventually undergo programmed cell death and thereby contribute to
sculpturing many organs and tissues [Meier, 2000]
• In human body about one lakh cells are produced every second by
mitosis and a similar number die by apoptosis (Vaux and Korsmayer
,1999, cell)
• Between 50 and 70 billion cells die each day due to apoptosis in the
average human adult. For an average child between the ages of 8 and
14, approximately 20 billion to 30 billion cells die a day. ( Karam, Jose A.
(2009). Apoptosis in Carcinogenesis and Chemotherapy. Netherlands: Springer.
ISBN 978-1-4020-9597-9)
• Without apoptosis, human gut can grow up to 12 miles in length
• Whole epithelial lining in our body changes every 23 days

11. sclero dinesh
APOPTOSIS NECROSIS
NATURAL YES NO
EFFECTS BENEFICIAL DETRIMENTAL
Physiological or
pathological
Always pathological
Single cells Sheets of cells
Energy dependent Energy independent
Cell shrinkage Cell swelling
Membrane integrity
maintained
Membrane integrity lost

12. sclero dinesh
APOPTOSIS NECROSIS
Role for mitochondria and cytochrome C No role for mitochondria
No leak of lysosomal enzymes Leak of lysosomal enzymes
Characteristic nuclear changes Nuclei lost
Apoptotic bodies form Do not form
DNA cleavage No DNA cleavage
Activation of specific proteases No activation
Regulatable process Not regulated
Evolutionarily conserved Not conserved
Dead cells ingested by neighboring cells Dead cells ingested by neutrophils and
macrophages

13. WHY APOPTOSIS?
To eliminate cells that :
• are potentially harmful
• have outlived their usefulness / aged
• are damaged beyond repair

14. • Since it is genetically regulated , apoptosis is
sometimes referred to as programmed cell death.
• Certain forms of necrosis , called “ necroptosis”
are also genetically programmed, but by a distinct
cell of genes.

15. MECHANISM OF APOPTOSIS
2 phases of apoptosis
1. Initiation phase - activate the caspases
2 pathways:
 Intrinsic / mitochondrial pathway – caspase 9
 Extrinsic / receptor mediated pathway - 8
2. Execution phase – caspases causes degradation

16. INTRINSIC PATHWAY
( MITOCHONDRIAL PATHWAY )
INTRINSIC PATHWAY
[ MITOCHONDRIAL PATHWAY ]

17. PLAYERS
1) Sensors – BAD , BIM , BID
2) Proapoptotic- BAX , BAK
3) Cytochrome C
4) Apaf-1 [ apoptosis activating
factor – 1 ]
5) Caspases
DEFENDERS
1) Antiapoptotic -
BCL 2,
BCL XL,
2) IAPS: [ inhibitors of
apoptosis

18. 1.Caspases:
• ‘c’ - cysteine protease(an enzyme with cysteine in its
active site)
‘aspase’- ability of these enzyme to cleave after aspartic
acid residues
• More than 10 members
• Central executioners of cell death
• Depending on order in which they are activated during
apoptosis, they are divided into
• Initiator caspase - 8, 9
• Executioner caspase -3, 6,7
 Presence of cleaved,active caspases is a marker for cells
undergoing apoptosis

19. 2.Bcl-2 proteins:
Family of more than 20 members - categorised in 3 groups
[ based on proapoptotic and antiapoptotic properties ,
and presence of BCL-2 homology (BH) domains ]
1) Antiapoptotic - BCL 2, BCL XL
• present on outer mito.membrane, cytosol and ER
Functn : make outer mitochondrial membrane
impermeable and prevent leakage of cytochrome
c.
Stimulated by : growth factors , survival signals
BCL XL - BCL2 related protein, long isoform

20. 2) Proapoptotic – BAX , BAK
Funtn : promote outer mito. Membrane
permeability by forming a channel and
promote leakage of cytochrome c
3) Sensors – BAD , BIM , BID
• Act as sensors of cellular stress and
damage
• Regulate other two groups
BAX – BCL2 associated X protein,
BAK- BCL2 antagonist killer 1,
BAD- BCL2 antagonist of cell death,
BID – BH3 interacting domain death
agonist

21. 3. Cytochrome C
– Mitochondrial protein present at inter
mitochondrial space
– Can activate caspase cascade
4. Apaf-1 [ apoptosis activating factor – 1 ]
– Present in cytosol
– Forms apoptosome after combining with
cytochrome c

22. 5 . IAPS: [ inhibitors of apoptosis ]
– Newly discovered group of anti-apoptotic proteins
present in cytosol
– 7 members identified-NAIP,cIAP-1,cIAP-2 and survivin
– Bind to and inactivate caspases
– Survivin is involved in spindle cell formation

23. INTRINSIC PATHWAY [MITOCHONDRIAL PATHWAY]
Withdrawal of Radiation , Toxins, Free radicals
survival signals
DNA damage / misfolded proteins
Loss of antiapoptotic
(BCL2, BCLXL )
function ER STRESS
activation of sensors of Bcl 2 family
activation of proapoptotics- BAX, BAK
oligomerization and increase in outer
mitochondrial membrane permeability
Leakage of cytochrome c leakage of SMAC which bind into
cytosol to neutralise IAP’s in cytosol

24. Contd……
Cytochrome c in cytosol + APAF 1
Form Hexamer ( Apoptosome )
Binds to caspase- 9 (
critical initiator caspase)
Enzyme cleaves the adjacent caspase 9 molecules
i.e . Autoamplification process
Execution phase

25. EXTRINSIC PATHWAY

26. Death Receptors
Members of TNF receptor family which contain a
cytoplasmic domain involved in protein-protein
interactions .i.e called Death Domain .
2 types of death receptors are there :
• TNFr (tumour necrosis factor receptor )
• FasR (fatty acid synthetase receptor )
The ligand for Fas is called FasL
The ligand for TNFr is TNFα
Adaptor Proteins
also contain death domain
a. FADD [ Fas associated death domain ]
b. TRADD [ TNF receptor associated death domain]

27. FAS ligand TNF
Death
domains
Adaptor proteins
Pro-caspase 8 (inactive)
Caspase 8 (active)
Pro-execution caspase
(inactive)
Execution caspase (active)
• Fas and the TNF receptor
are integral membrane
proteins with their receptor
domains exposed at the surface of
the cell
• Binding of the complementary
deathactivator (FasL and TNF resp
ectively) transmits a signal to the
cytoplasm
• FADD /TRADD attach to death receptors
• Binds an inactive form of caspase-8
• M/L procaspase 8 molecules are brought
• They cleave and generate active
caspase 8

28. • This pathway can be inhibited by a protein FLIP
which binds to pro –caspase 8 and cannot
cleave it
• Some viruses and normal cells produces FLIP
and use this inhibitor to protect themselves
from Fas mediated apoptosis
FLIP – FLICE inhibitory protein

29. • Extrinsic and intrinsic pathway involve
fundamentally different molecules for their
initiation, but their may be an
interconnection between them.
Eg : in hepatocytes and pancreatic β cells,
caspase 8 produced by Fas signaling cleaves
and activate sensors of bcl family i.e BID
which then stimulate mitochondrial
pathway

30. EXECUTION PHASE
• Final phase of apoptosis
• Mediated by proteolytic cascade
• After initiator caspases ( 2, 8, 9 and 10) are cleaved to
generate its active form, the enzymatic death program
is set in motion by rapid sequential activation of the
executioner caspases – 3,6
• Caspase -3 activates DNase which causes degradation
of chromosomal DNA within the nuclei and causes
chromatin condensation.
• Caspase -3 induces cytoskeletal reorganisation and
disintegration of cell into apoptotic bodies.

31. REMOVAL OF DEAD CELLS
Factors by which apoptotic cells attracts phagocytes
towards them :
• Apoptotic bodies- “bite size”-edible for phagocytes
• Phosphatidyl serine “flips” out from inner to outer
layer –recognized by macrophages receptors
• Some apoptotic bodies express thrombospondin
recognized by phagocytes
• They are coated by Ab and C1q

32. 32

33. MORPHOLOGY OF CELLS IN APOPTOSIS
• Cell Shrinkage and dense cytoplasm
• Chromatin condensation to periphery and later
fragmentation.
• Plasma membrane is intact
• Formation of cytoplasmic blebs and apoptotic
bodies.
• Structurally altered so that the apoptotic cell
becomes “tasty” for phagocytosis
• Phagocytosis of apoptotic bodies by macrophages
• Dead cell is rapidly cleared before contents are
leaked out
• No inflammatory reaction

34. MORPHOLOGY OF APOPTOSIS
Progressive cell shrinkage
Chromatin condensation
Plasma membrane blebbing
Apoptotic bodies
Phagocytosis - no inflammation

35. MORPHOLOGY OF CELLS IN APOPTOSIS

36. Various methods available for
detection of apoptotic cells
1) Light microscopy
• In H&E the apoptotic cell appears as round or
oval mass of intensely eosinophilic cytoplasm
with fragments of dense nuclear chromatin

38. MICROSCOPY
Apoptosis in neutrophils

39. Councilman bodies- liver tissue

40. 2) Electron microscopy
Defines subcellular changes
like
• Chromatin condensation
• Plasma membrane
blebbing

41. 3) Gel electrophoresis
DNA ladder pattern Single cell gel
electrophoresis comet assay
showing apoptotic cell

42. 4) TUNEL [ terminal deoxy transferase
mediated dUTP nick end labelling ]
• Identify DNA breaks in
apoptosis
Apoptotic cells detected by TUNEL and fluoresce green;
while necrotic cells are stained with red-fluorescent
propidium iodide

43. 5) Immunohistochemistry
Immunohistochemical
detection of apoptotic cells
using antibodies against a
wide range of substrates
most importantly :
• Caspase 3
• P53
• Annexin V

44. APOPTOSIS IN PHYSIOLOGIC
CONDITIONS
1) Programmed destruction of
cells during embryogenesis
including implantation,
organogenesis ,
developmental involution
and metamorphosis.
Eg : separation of webbed
fingers and toes in embryo

45. Apoptosis in bud
formation during
which many
interdigital cells
die. They are stained
black by a TUNEL
method
Incomplete differentiation
in two toes due to lack of
apoptosis
sclero dinesh

46. 2) Involution of hormone-dependent tissues upon
hormone withdrawal…
Eg :
• Endometrial cell breakdown during menstrual
cycle
• Ovarian follicular atresia in menopause
• Regression of the lactating breast after weaning
• Prostatic atrophy after castration

47. 3) Cell loss in proliferating cell
populations
Eg :
• Immature lymphocytes in bone
marrow and thymus that fail to
express useful antigen receptors
• B lymphocytes in germinal centers
• Epithelial cells in intestinal crypts
So as to maintain a constant number…

48. 4) Elimination of potentially harmful self-reactive
lymphocytes
5) Death of host cells that have served their useful
purpose
Eg :
- Neutrophils in an a/c inflammatory response
- Lymphocytes at the end of an immune response
In these situations , cells undergo apoptosis
because they are deprived of necessary survival
signals such as growth factors.

49. Apoptosis: Role in Disease
TOO MUCH: Tissue atrophy
TOO LITTLE: Hyperplasia
Neurodegeneration
Thin skin
etc
Cancer
Athersclerosis
etc

50. APOPTOSIS IN PATHOLOGIC
CONDITIONS
Pathological condition arise as a result of
dyregulation in apoptosis……………….
i.e defective apoptosis with increase cell survival
OR
Increased apoptosis with increased cell death

51. DYSREGULATED APOPTOSIS
DISORDERS WITH DEFECTIVE
APOPTOSIS AND INCREASE CELL
SURVIVAL
Eg :
• Mutations of p53 -------------
CANCERS
• AUTOIMMUNE DISEASES
DISORDERS WITH INCREASED
APOPTOSIS AND EXCESS CELL
DEATH
Eg :
• NEURODENEGERATIVE D/S
• Ischemic injury
• Death of virus infected cells. Eg
-AIDS

52. Apoptosis: Role in Disease
Aging
Aging --> both too much and too little apoptosis
(evidence for both)
Too much (accumulated oxidative damage?)
---> tissue degeneration
Too little (defective sensors, signals?
---> dysfunctional cells accumulate
hyperplasia (precancerous lesions)
sclero dinesh

53. PREGNANCY ASSOCIATED DISEASE AND APOPTOSIS
• During pregnancy trophoblast cells from placenta
invade the uterine environment inorder to remodel
the maternal blood vessels and help establishing and
maintain sucessful pregnancy.
• Strict control over the cell proliferation and
apoptosis is required to achieve this.
• In some cases the process can be compromised
and excessive apoptosis of trophoblast cells 
failure of fully remodel the maternal environment
 complication of pregnancy
Eg : preeclampsia of pregnancy

54. AUTOIMMUNE D/S AND APOPTOSIS
IN HEALTHY BODY
• T and B – lymphocytes are cells of immune system that
are responsible for destroying infected or damaged cell
in the body.
• They mature in thymus, but before they can enter the
blood stream they are tested to ensure that they are
effective against foreign antigen and are also not
reactive against normal healthy cells.
• Any ineffective or self reactive T-cells are removed
through induction of apoptosis.

55. AUTOIMMUNE D/S AND APOPTOSIS ….contd
IN DISEASE :
• Poor regulation of apoptosis in T- lymphocyte results
Autoimmune D/s of cytotoxic T lymphocytes.
Eg : Behcet’s D/S, Ankylosing Spondylitis, SLE
• Poor regulation of apoptosis in B –cell
Eg : SLE , Scleroderma , Multiple sclerosis
• RHEUMATOID ARTHRITIS : excessive proliferation of
synovial cells is thought to be due in part to the
resistence of these cells to apoptosis.
• AUTOIMMUNE LYMPHOPROLIFERATIVE SYNDROME [ALPS]
: mutation in FAS gene

56. Neurodegenerative D/S and Apoptosis
IN HEALTHY BODY :
• During development of central and peripheral nervous
system, many neurons undergo apoptosis that coincides
with synaptogenesis.
• Signals that determine whether or not developing neurons
live or die may include competition for a limited supply of
target derived neurotrophic factors and activation of
receptors for excitatory neurotransmittor Glutamate.
• Initial overproduction of neurons followed by death of
some is an adaptive process that provide enough neurons
to form nerve cell circuits.

57. Neurodenerative D/S and Apoptosis…contd
Metabolic stress [ stroke, aging ]
Oxidative stress and free radicals DNA damage
Inherited mutations misfolded proteins
ER stress
APOPTOSIS increase Ca influx

58. Neurodenerative D/S and Apoptosis…contd
IN DISEASE :
• apoptosis of hippocampal neuron  Alzheimer’s D/s
Over expression of Bcl 2
• apoptosis of midbrain neurons that uses NT’s
dopamine  Parkinson’s Disease
• apoptosis of neurons in striatum which control body
movements  Huntington’s Disease
• apoptosis of lower motor neurons  Amyotrophic
Lateral Sclerosis
• In stroke  activation of glutamate receptors which
act as a trigger to stimulate apoptosis

59. Apoptosis: Role in Disease
Cancer
• Apoptosis eliminates damaged cells (damage => mutations =>
cancer)
• Apoptosis is regulated by two major genes p53 & Bcl-2.
• Tumor suppressor p53 controls senescence and apoptosis
responses to damage.
• Mutations or overexpression of these genes will result in Cancer.
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 ===> CANCER

60. Apoptosis: Role in Disease
Cancer
Virus associated cancer
•Several human papilloma viruses (HPV) have been implicated
in causing cervical cancer. One of them produces a protein (E6)
that binds and inactivates the apoptosis promoter p53.
•Epstein-Barr Virus (EBV), the cause of mononucleosis and
associated with some lymphomas
– produces a protein similar to Bcl-2
– produces another protein that causes the cell to increase its
own production of Bcl-2. Both these actions make the cell
more resistant to apoptosis (thus enabling a cancer cell to
continue to proliferate).

61. • Some B-cell leukemia and lymphomas express high
levels of Bcl-2, thus blocking apoptotic signals they may
receive.
• Melanoma (the most dangerous type of skin cancer)
cells avoid apoptosis by inhibiting the expression of the
gene encoding Apaf-1.
Apoptosis: Role in Disease
Cancer

62. •Other cancer cells express high levels of FasL, and can kill any
cytotoxic T cells (CTL) that try to kill them because CTL also
express Fas (but are protected from their own FasL).
•Some cancer cells, especially lung and colon cancer cells,
secrete elevated levels of a soluble "decoy" molecule that
binds to FasL, plugging it up so it cannot bind Fas. Thus,
cytotoxic T cells (CTL) cannot kill the cancer cells
Apoptosis: Role in Disease
Cancer

63. – Cancer cells
• Radiation and chemicals used in cancer therapy induce apoptosis
in some types of cancer cells.
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Fig. 1: induced apoptosis in stomach carcinoma cells
Left: Before induction
Middle: 24h after induction
Right: 48h after induction

64. • Apoptosis and AIDS
Human Immunodeficiency Virus infects CD4+
T cells and HIV Tat protein increases the
expression of Fas receptor, resulting in
excessive apoptosis of T cells.
Hallmark- the decline in the number of the
patient's CD4+ T cells (normally about 1000
per microliter (µl) of blood).
sclero dinesh

65. ANTI-APOPTOTIC THERAPY IN DISEASES
• Stimulation of IAP - for the treatment of stroke,
spinalcord injuries , multiple sclerosis
• Synthetic nonspecific caspase inhibitors – trials going on
treatment of myocardial reperfusion injury, RA
• Aim of the treatment in neurodegenerative d/s is to
block apoptotic triggers and activation of anti -
apoptotic pathway ( by neurotrophic factors )
Eg : block amyloid β production  t/t Alzheimer’s d/s
block glutamate receptor activation  stroke
using neurotrophic factors like
vitamin E  t/t alzheimer’s
Insulin like Growth factors  t/t ALS

66. CONCLUSIONS
• Apoptosis is regarded as a carefully regulated energy dependent
process, characterized by specific morphological and biochemical
features in which caspase activation plays a central role.
• The importance of understanding the mechanistic machinery of
apoptosis is vital because programmed cell death is a component
of both health and disease, being initiated by various physiologic
and pathologic stimuli.
• Moreover, the widespread involvement of apoptosis in the
pathophysiology of disease lends itself to therapeutic
intervention at many different checkpoints.
• Understanding the mechanisms of apoptosis, and other variants
of programmed cell death, at the molecular level provides deeper
insight into various disease processes and may thus influence
therapeutic strategy.

67. References
• Robbins and Cotran,pathologic basis of disease, Kumar et al, 9th
edi.,2014
• Henry’s clinical diagnosis and management,21st edi,2007,546
• Review article on apoptosis, Indian Journal of Cancer ,vol 35, No.4,
sep- 2007 , 495-516
• Review article on various method available for detection of apoptotic
cell, Indian Journal of Cancer, july-sep 2013 , vol 50, issue 3.
• www.ncbi.nlm.nih.gov
• www.reading.ac.uk

68. THANK YOU