This presentation is about Necrosis, Apoptosis and Necroptosis in physiologic and some pathological conditions

2.  A German scientist: Carl Vogt(1842)
 Walther Fleming(1885)
 John Foxton Ross Kerr(1965)
 Kerr received Ehrlich price for full
description(2000)

3. Cells are born, live for a given
period of time and then die
Bowen, 1998
--- Physiological cell death
--- Cell suicide
--- Cell deletion
--- Programmed cell death
-- Or Depilatory!..
APOPTOSIS
Cells are born, live for
a given period
of time and then die

4. ... Growth of Embryo
... Tissue Homeostasis
... Immunology
... Chronic viral diseases
... Neurodegenerative diseases
... Reperfusion injury
... Insuline-dependent Diabetes
... Atherosclerosis
... Myocard Infarction
... AIDS
... Development and Treatment of Malignancies

5.  Apoptosis is needed for proper development
› 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
› Autoreactive immune cells
› Cells with DNA damage
› Cancer cells

6.  Apoptosis plays an important role
in normal development and homeostasis*
 Cancer is often initiated by DNA damage
 Normal cells undergo apoptosis in response to
stress-inducing events in the cell, such as DNA damage
 Dysregulation of apoptosis is critical for cancer
development and tumor cell survival
*A property of cells, tissues, and organisms that allows the maintenance and
regulation of the stability and constancy needed to function properly

7. 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

8.  Withdrawal of positive (Growth) signals
› Nerve Growth Factors(NGF)
› Interleukin-2 (IL-2)
 Receipt of negative (Death) 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)

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

10.  Cell death by injury
-Mechanical damage
-Exposure to toxic chemicals
 Cell death by suicide
 Internal signals
 p53
 External signals
 Toxins, hormones, growth factors, NO &
cytokines

11. MAJOR PLAYERS IN APOPTOSIS
 Caspases
 Death signals e.g. TNF & TNFR
 p53
 BAX
 Bcl-2 family

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

15. Aspartate- AA
proteolysis
Nuclear
BAX (proapoptotic)
Cyt C
Apaf-1
p53DNA damage
Mitochondrial
Initiator
Caspase-8E / 9I
Effector
Caspase- 3
CAD
(DNA-ase)
DNA nucleosomal
200 bp fragments
bcl- 2
Anti-apoptotic
p21
Cell Cycle
Arrest
DNA
Repair

17. https://www.youtube.com/watch?v=1vaEVcMfa1E
https://www.youtube.com/watch?v=-vmtK-bAC5E

18. Apoptosis
Caspases
Pro-apoptotic receptor
Pro-apoptotic ligand
Cell-extrinsic
pathway
Cell-intrinsic
pathway
BAX
BCL2
PUMA
Stress
Mitochondria
BAX, BCL2-associated protein; BCL2, B-cell chronic lymphocytic leukemia/lymphoma 2; PUMA, p53-
upregulated modulator of apoptosis.

19. APAF1, apoptotic protease activating factor-1; BAK, BCL2 homologous antagonist/killer; BAX, BCL2-associated protein; BCL2, B-cell chronic
lymphocytic leukemia/lymphoma 2; BCLXL, BCL2-like 1; IAP, inhibitor of apoptosis protein; MCL1, myeloid cell leukemia sequence 1 (BCL2-
related); PUMA, p53-upregulated modulator of apoptosis; SMAC/DIABLO: second mitochondria-derived activator of caspase/direct IAP binding
protein with low pI.
Chemotherapy
Radiotherapy
Caspase 3, 6, 7
Apoptosis
p53
BAX, BAK
Cell-intrinsic
pathway
BCL2, BCLXL,
MCL1
Mitochondria
SMAC/DIABLO
p53
Caspase 9
DNA damage
PUMA, NOXA
p53
APAF1
Cytochrome c
DNA damage
IAP

20. Initiator caspases
Pro-apoptotic stimulus
Effector caspases
Caspase
cascade
Apoptosis
Adapted from Thornberry NA, Lazebnik Y. Science 1998;281:1312–1316.
Caspase, cysteine aspartase.

21. CELL SURFACE DEATH RECEPTORS

22.  The intrinsic pathway is triggered by the p53 tumor-suppressor in
response to DNA damage and other types of severe cell stress
 Conventional anticancer therapies, such as chemotherapy and
radiotherapy, activate this pathway via p53
 p53 activates the intrinsic pathway through transcriptional upregulation of
pro-apoptotic members of the BCL2 family of proteins such as PUMA and
BAX
 BAX causes the release of cytochrome c from the mitochondria, which
together with the adaptor APAF1, activate the initiator caspase 9
 Caspase 9 activates the effector caspases 3, 6, and 7, which are
responsible for destroying critical components of the cell, and inducing
apoptosis
 p53 is inactivated by mutations in more than half of human cancers

23.  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.

26. https://www.youtube.com/watch?v=4wPlw_Bdz7Q

27. Necroptosis can be initiated by TNFα, TLR agonists, ultraviolet
light, ionizing radiation, reactive oxygen species……

28.  DEREGULATION OF APOPTOSIS IN
MALIGNANCIES
 POTENTIAL ROLE OF APOPTOSIS IN
CANCER TREATMENT

29. 1) A number of activities take place

30. 2) Translocation of
phosphatidylserine
3) ATP-dependency
4) Internucleosomal DNA
fragmentation (ladder pattern)
5) No apoptosis at +4 oC
6) No inflammation

31. Adapted from Ashkenazi A. Nat Rev Cancer 2002;2:420–430.
FADD, Fas-associated death domain; FLIP, FLICE (FADD-like interleukin 1β-converting enzyme) inhibitory protein.
Pro-apoptotic ligand
Caspase 3, 6, 7
Apoptosis
FADD
FLIP
DR5
DR4
Cell-extrinsic
pathway
Procaspase 8, 10
Caspase 8, 10

32.  The extrinsic pathway triggers apoptosis in response to the
activation of pro-apoptotic receptors, such as DR4 and DR5,
by specific pro-apoptotic ligands, such as Apo2L/TRAIL
 This pathway stimulates apoptosis independently of p53
 Ligand-induced activation of DR4 and DR5 leads to the rapid
assembly of the death-inducing signaling complex (DISC)
and the recruitment of initiator caspases 8 and 10 through
the adaptor Fas-associated death domain (FADD)
 Caspases 8 and 10 activate effector caspases 3, 6, and 7,
leading to apoptosis

33. Adapted from Ashkenazi A. Nat Rev Can 2002;2:420–430.
APAF1, apoptotic protease activating factor-1; BAK, BCL2 homologous antagonist/killer; BAX, BCL2-associated protein; BCL2,
B-cell chronic lymphocytic leukemia/lymphoma 2; BCLXL, BCL2-like 1; BID, BH3-interacting domain death agonist; DR, death receptor;
FADD, Fas-associated death domain; FLIP, FLICE (FADD-like interleukin 1β-converting enzyme) inhibitory protein; IAP, inhibitor of apoptosis
protein; MCL1, myeloid cell leukemia sequence 1 (BCL2-related); PUMA, p53-upregulated modulator of apoptosis; SMAC/DIABLO: second
mitochondria-derived activator of caspase/direct IAP binding protein with low pI.
Caspase 3, 6, 7
Apoptosis
Pro-apoptotic ligand
FADD
FLIP
DR5
DR4
Cell-extrinsic
pathway
Procaspase 8, 10
p53p53
Caspase 9
Caspase 8, 10
p53
BAX, BAK
Mitochondria
SMAC/DIABL
O
Chemotherapy
Radiotherapy
DNA damage
PUMA, NOXA
APAF1
Cytochrome c
DNA damage
BID
IAP
Cell-intrinsic
pathway
BCL2, BCLXL,
MCL1

34. Endogenous
Apo2L/TRAIL
DR4 DR5
Apo2L/TRAIL, apoptosis-inducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligand; DR, death receptor.

35. DR4 DR5
DR, death receptor.

36. FADD
DR4 DR5
DR, death receptor; FADD, Fas-associated death domain.

37. DR4 DR5
Procaspase 8, 10 Procaspase 8, 10
DISC
DR, death receptor; DISC, death-inducing signaling complex.

38. Caspase 8,10 Caspase 8,10
DR4 DR5
DR, death receptor.

39. DR4 DR5
Caspase 3, 6, 7 Caspase 3, 6, 7
Caspase 8, 10 Caspase 8, 10
DR, death receptor.

41. SUBSTRATES for CASPASES
... PARP
... DNA-PK
... pRb
... Lamins
... NuMA
... Fodrin
... -Aktin
... Mdm2
... Cyclin A2
... Presenilin
... Others

42. THE APOPTOTIC PATHWAY
Triggers Modulators Effectors Substrates DEATH
. FADD
. TRADD
. FLIP
. Bcl-2 family
. Cytochrome c
. p53
. Mdm2
. Caspases . Many cellular
proteins
. DNA
. Growth factor
Deprivation
. Hypoxia
. Loss of adhesion
. Death receptors
. Radiation
. Chemotherapy

43. AN APOPTOTIC CELL IN CULTURE

45. Mitochondria-mediated caspase activation at the apoptosome.
A. Apoptotic stimuli trigger the release of apoptogenic factors from the mitochondrial intermembrane
space to the cytosol, such as cytochrome c which induces the formation of the apoptosome and the
activation of procaspase-9. B. By the action of cytochrome c (Cyto C) and dATP the Apaf-1 protein
adopts a conformation that allows the formation of a heptameric, wheel-like structure, the apoptosome.
Procaspase-9 molecules can bind to the inner “hub” region of the apoptosome and are activated by
dimer formation. Active caspase-9 dimers further mediate activation of effector caspases

46. From the archive of Dr Ulukaya

48. Transfection studies in rat fibroblasts
Ras
Apoptosis
Tumor growth
c-myc
Apoptosis
Tumor growth
1

49. Igney and
Krammer
1999
2

50. 3

51. APOPTOSIS-RELATED CELLULAR
PROTEINS INVOLVE IN THE PROGRESSION
OF MALIGNANCIES
... p53
... pRb
... Fas
... Mdm2
... c-myc
... c-Jun
... Bcl-2 family
4

52. Bcl-2 FAMILY
- Bcl-2
- Bcl-XL
- Mcl-1
*******************
- p35 (Baculovirüs)
-BHRF1 (Ebstein-Barr
Virüsü)
- LMW5 HL (“African Swine
Fever Virus”)
- p19 (E1B) (Adenovirüs)
- Bax
- Bcl-XS
- Bak
- Bad
***************
-????
Anti-apoptotic Pro-apoptotic

53. Bcl-XL
Bad
Bcl-XL
Bax
Bcl-2
Bax
Bax Bax
Bcl-2
Bad
CELL
SURVIVAL
CELL DEATH

54. Various Expression Levels of Apoptosis-Related
Proteins Determine Patient-Specific Malignancy ?
5

55.  "Right now we lump
patients together and
treat them with the
same drugs and then
deal with their
variable response to
treatment. We're
essentially treating
different diseases
with the same
medicine.”
 Richard Klausner,
1997OncoTech, Inc

56. Question 1 ...

57. Question 2 ...

58. POTENTIAL ROLE OF APOPTOSIS IN
CANCER TREATMENT

59. Things to do ....
Determination of the Apoptosis-
Related Proteins
(1)

60. . p53 gene status--------------- Modulates the chemosensitivity
. p53 level –---------- Predictor for the response to chemo- or
radiotherapy (Advanced Head and Neck Carcinomas,
Epithelial Ovarian Ca)
. Mutant p53 --------- Overall shortened survival (Breast Ca)
. Ratio of Bcl-2/Bax -----------------------–--- Prognostic factor
(Hematologic Malignancies, Colon Ca)
. Bcl-2 alone –-------- Prognostic factor (Advanced Over Ca)

61. Measurement of the Cytotoxic (Apoptosis-
Inducing) Effects of Chemotherapeutic
Agents on Individual Cancer Tissue
Specimens Removed from Cancer Patients
Things to do ....
(2)

62.  Designation of Patient-Specific
Chemotherapy

65. From the archive of Dr Ulukaya

66. ATP-Tumour Chemosensitivity Assay
Tumour
1mm3 Fragments
Overnight enzyme
dissociation
Wash cells,
count and
estimate viability
Plate at 20,000
cell/well
Incubate for 5-7 days,
extract ATP and read
in a luminometer
Kindly supplied from Dr I Cree

67. In the literature (1)....

68. ... Retrospective clinical correlation in breast carcinoma (Cree
et al, 1996): 97% assay evaluability, 76% accuracy, 27%
imrovement in clinical response rate
... A greater benefit with regard to both ORR and PFS in
platinum refractory patients (Kurbacher et al, 1998): Overall
survival, 97 weeks / 69 weeks; Response rate, 64% / 37%

70.  It is considered that defective apoptosis is a feature of
malignant development
 Induction of apoptosis in malignancies is to be aimed
 Detection of apoptosis-related proteins may be of
importance in the prediction of patient’s response to
chemo- or radio-therapy as well as of survival rates
 Chemosensitivity testing, thereby individualised
chemotherapy on the basis of patient-specificity, seems to
be promising in the succesful treatment of malignancies.
This testing, thereby, may revolutionize the way we use
anti-cancer drugs in near future