MEDICAL COUNCIL OF INDIA, COMPETENCY BASED UNDERGRADUATE CURRICULUM FOR THE INDIAN MEDICAL GRADUATE, 2018

1. PA 2.4
DESCRIBE AND DISCUSS CELL DEATH, TYPE &
MECHANISM, NECROSIS, APOPTOSIS,
DIFFERENCES BETWEEN NECROSIS & APOPTOSIS
AUTOLYSIS
Dr IRA BHARADWAJ
MCI TEACHER ID: PAT 2300569
KUHS FACULTY ID: M21512

2. TEXTBOOK REFRENCES
• ROBBINS BASIC PATHOLOGY
• HARSH MOHAN TEXTBOOK OF PATHOLOGY
• OTHER STANDARD REFRENCES

3. SLO
• TYPES OF CELL DEATH
• DEFINITION OF APOPTOSIS
• ETIOLOGY OF APOPTOSIS
• PATHOGENESIS OF APOPTOSIS
• BIOCHEMICAL CHANGES IN APOPTOSIS
• LAB DX OF APOPTOSIS

4. SLO
• EXAMPLES OF APOPTOSIS
• MORPHOLOGY OF APOPTOSIS
• DIFFERNCES BETWEEN NECROSIS &
APOPTOSIS
• AUTOLYSIS

5. CELL DEATH
TYPES
• NECROSIS [already discussed]
• APOPTOSIS
• AUTOLYSIS

7. APOPTOSIS
DEFINITION
Regulated (programmed) cell death, to
remove unwanted & irreparably
damaged cells with minimal host
reaction
There is no leakage of cell contents.
Activated cellular enzymes degrade nuclear
& cytoplasmic proteins resulting in
fragmentation of cell contents enclosed

8. APOPTOSIS
DEFINITION
by intact but biochemically altered cell
membrane to form apoptotic bodies
which are rapidly phagocytosed without
inflammation
Necrosis & apoptosis may coexist -
necroptosis

9. APOPTOSIS
ETIOLOGY
PHYISIOLOGICAL – removal of cells at the end of
life span & limit no of cells in proliferating
tissues.
• Embryogenesis (programmed cell death)
• Involution of hormone dependent tissues eg
menstruation, post lactation breast
• Cell loss in proliferating tissues eg, bone
marrow

10. APOPTOSIS
ETIOLOGY
• Elimination of cells after their use is over
eg neutrophils at end of inflammation,
lymphocytes at end of immune response
• Elimination of self reactive lymphocytes,
• Cytotoxic T lymphocytes kill cells by
apoptosis

11. APOPTOSIS
ETIOLOGY
PATHOLOGICAL
• Remove genetically / irreparably
damaged cells without inflammation
• Cell injury in viral infections ( HIV )
• Apoptosis of cells in organ with
slowly developing duct obstruction

12. APOPTOSIS
ETIOLOGY
• DNA damage by radiation, drugs,
hypoxia, temperature extremes may
damage cells directly / ROS to induce
apoptosis/necrosis
• Excess of misfolded proteins leading to
ER stress

13. APOPTOSIS
PATHOGENESIS
Activation of enzymes – caspases
(cystine proteases that cleave proteins
after aspartic residue) by
• Mitochondrial (intrinsic) pathway
• Death receptor (extrinsic) pathway
• Common pathway

14. APOPTOSIS
PATHOGENESIS
OTHER MECHANISMS OF APOPTOSIS:
• Calcium directly activates caspases &
increases mitochondrial permeability,
• ROS – protein & DNA damage
• CYTOTOXIC T LYMPHOCYTES- secrete
granzymes which directly activate
executioner caspase [common pathway]

15. PATHOGENESIS OF APOPTOSIS
MITOCHONDRIAL (INTRINSIC)PATHWAY
Bcl-2 family (about 20 proteins)
• Sensor proteins - BH3 activated by
etiology
• Proapoptotic proteins –Bak, Bax
increased
• Anti apoptotic proteins – Bcl-2, Bcl-
x1 decreased

16. PATHOGENESIS OF APOPTOSIS
MITOCHONDRIAL (INTRINSIC)PATHWAY
Bak & Bax - affects permeability of
mitochondrial membrane – to form channels
for escape of:
• Proteins: proapoptotic & inhibitors of anti
apoptotic factors in cytoplasm
• Cytochrome C: activate caspase 9 (initiator)
Mitochondrial pathway is the commonest
mechanism of apoptosis.
Mitochondria have important role in both
necrosis & apoptosis

18. PATHOGENESIS OF APOPTOSIS
DEATH RECEPTOR ( EXTRINSIC ) PATHWAY
• Cell surface has Death Receptors for TNF &
Fas (CD95)
• FasL present on activated TL & cytotoxic T
lymphocytes bind to Fas on cell membrane
• Activate caspase 8 (initiator)
• Activates BcL2 family (proapoptotic - Bid )
leading to apoptosis via mitochondrial
pathway also

19. PATHOGENESIS OF APOPTOSIS
DEATH RECEPTOR ( EXTRINSIC ) PATHWAY
• FLIP – anti caspase, blocks DR pathway,
viruses produce FLIP like chemical to
ensure cell survival of infected cells
• Death receptor pathway is used for
removal of cells by cytotoxic TL & self
reactive TL

20. PATHOGENESIS OF APOPTOSIS
COMMON PATHWAY
• Caspase 8 & 9 ( INITIATOR CASPASES )
• Activate caspase 3&6 ( EXECUTIONER
CASPASES ) , these
• Cleave DNA, nucleoproteins and
cytoskeleton, resulting in
• Fragmentation of cells
• Each fragment is surrounded by cell
membrane & is ka apoptotic body

22. PATHOGENESIS OF APOPTOSIS
APOPTOTIC BODIES
• Apoptotic bodies are degraded contents
of cells, bound by cell membrane
• Biochemical changes in cell membrane
induce phagocytosis by tissue
macrophages & other phagocytic cells
without inflammation

23. PATHOGENESIS OF APOPTOSIS
CLEARANCE OF APOPTOTIC BODIES
• In normal cells phosphatidylserine is present
on the inner leaflet of the plasma membrane,
but in apoptotic cells this phospholipid "flips"
out and is expressed on the outer layer of the
membrane, where it is recognized by
macrophages.
• Cells that are dying by apoptosis also secrete
soluble factors that recruit phagocytes

24. BIOCHEMICAL CHANGES IN APOPTOSIS
• Proteolysis of cytoskeleton due to breakdown
of protein cross linkages
• Fragmentation of nucleus by nuclease enzyme
• Flipping of phosphatidylserine from inner
surface of cell membrane to outer surface of
membrane , this attracts phagocytic cells
• In some cases trombospondin appears on
surface of apoptotic bodies , this attracts
phagocytes

25. RESEARCH LAB Dx OF APOPTOSIS
• ANNEXIN V ASSAY – this dye binds to
phosphatidylserine
• CHROMATIN CONDENSATION – seen in H&E stain &
other special nuclear stains like Feulgen , and
fluorescent stain (acridine orange)
• AGROSE GEL ELECTROPHORESIS – fragmented DNA
shows step ladder pattern
• ACTIVATED CASPASES & CYTOCHROME C ASSAY
• FLOW CYTOMETRY – shows rapid shrinkage of cells

26. APOPTOSIS
EXAMPLES
• GROWTH FACTOR DEPRIVATION
• DNA DAMAGE
• ACCUMALATION OF MISFOLDED PROTEINS (
ER STRESS )
• IMMUNE SYSTEM :
Self reactive lymphocytes
Cytotoxic t lymphocytes

27. APOPTOSIS
GROWTH FACTOR DEPRIVATION
SEEN IN
• Hormone dependent cells
• Lymphocytes not stimulated by antigens
• Decrease /absence of growth factors
• APOPTOSIS BY MITOCHONDRIAL PATHWAY –
increase proapoptotic factors & decrease anti
apoptotic factors

28. APOPTOSIS
DNA DAMAGE
• Exposure of cells to radiation , chemicals
[chemotherapeutic agents] & other causes
induces DNA damage
• When DNA is damaged, the p53 protein
accumulates in cells.
• p53 first arrests the cell cycle (at the G1
phase) to allow for repair of DNA
• If DNA is repaired cell returns to normal state

29. APOPTOSIS
DNA DAMAGE
• If DNA is not repaired, p53 triggers
apoptosis, mainly by stimulating
synthesis of pro-apoptotic Bcl-2 proteins
• Apoptosis occurs via mitochondrial
pathway

30. APOPTOSIS
DNA DAMAGE
• When p53 is mutated or absent (as it is
in certain cancers), it is incapable of
inducing apoptosis, so that cells with
damaged DNA are allowed to survive.
• In such cells, the DNA damage may result
in mutations or translocations that lead
to neoplastic transformation [cancer]

31. APOPTOSIS: ER STRESS
ACCUMULATION OF MISFOLDED PROTEINS
• Normal protein synthesis requires
folding of proteins in a specific
manner by chaperons in ER
• Misfolded proteins are removed by
ubiquitin – protease pathway

32. APOPTOSIS: ER STRESS
ACCUMULATION OF MISFOLDED PROTEINS
Accumulation of misfolded proteins in ER occurs
in genetic & acquired conditions
GENETIC CONDITIONS
• Cystic fibrosis,
• Familial hypercholesterolemia,
• A-1-AT deficiency

33. APOPTOSIS: ER STRESS
ACCUMULATION OF MISFOLDED PROTEINS
ACQUIRED [ENVIRONMENTAL] CONDITIONS
• aging
• mutations
• chronic nutritional deficiency
• oxygen deficiency
• infections
Present in Neurodegenerative diseases like
Alzheimer & Parkinson disease

34. APOPTOSIS: ER STRESS
ACCUMULATION OF MISFOLDED PROTEINS
• This leads to decrease in protein synthesis &
increase in chaperon synthesis resulting in
decreased levels of misfolded proteins
• When these adaptations fail, misfolded
proteins accumulate in ER leading to ER
stress
• ER stress activates caspases leading to
apoptosis via mitochondrial pathway

35. APOPTOSIS: IMMUNOLOGY
Self-Reactive Lymphocytes
• Lymphocytes capable of recognizing self
antigens are normally produced in all
individuals.
• If these lymphocytes encounter self
antigens, they (lymphocytes) die by
apoptosis via death receptor pathway
[CD95/ Fas]

36. APOPTOSIS: IMMUNOLOGY
Cytotoxic T Lymphocyte
• Cytotoxic T lymphocytes (CTLs)
recognize foreign antigens presented
on the surface of infected host cells
and tumor cells.
• Upon activation, CTL granule
proteases called granzymes enter
the target cells

37. APOPTOSIS: IMMUNOLOGY
Cytotoxic T Lymphocyte
• Granzymes cleave proteins at aspartate
residues and are able to activate cellular
caspases.
• In this way, the CTL kills target cells by
directly inducing the effector phase of
apoptosis, without engaging
mitochondria or death receptors.

38. MORPHOLOGY 0F APOPTOSIS
• Cell shrinkage
• Chromatin
condensation
• Formation of
cytoplasmic blebs
and apoptotic bodies
• Phagocytosis of
apoptotic cells or cell
bodies usually by
macrophages.

40. NECROSIS Vs APOPTOSIS
ETIOLOGY PATHOLOGICAL PHYSIOLOGICAL AS WELL
AS PATHOLOGICAL
PATHOGENESIS CELL MEMBRANE DAMAGE
RESULTING IN LEAKAGE OF
CONTENTS, does not
require energy
DEGRADATION OF
CELLULAR CONTENTS WITH
FORMATION OF APOPTOTIC
BODIES, requires energy
MORPHOLOGY 5 TYPES DEPENDING ON
ETIOLOGY, LIKE CASEOUS,
COAGULATIVE, OTHERS
FORMATION OF APOPTOTIC
BODIES
No of cells effected
Cell size
Cell membrane
Nucleus
Cellular contents
several
Increased
Broken
Pyknosis, karyorrhexis,
karyolysis
Digested , leak out
single
Decreased
Intact
Fragmented
Intact, may be degraded, do
not leak out
TISSUE REACTION INFLAMMATION NO INFLAMMATION

41. AUTOLYSIS
• Autolysis means self digestion
• Destruction of cell by self enzymes, usually
lysosomal
• Usually this process occurs in injured & dying
cells

42. ESSAY QS
• Write the causes, bio chemical features,
mechanisms and examples of apoptosis .
Differentiate between Necrosis and Apoptosis