Apoptosis

1. Apoptosis

2. Literal sense

3. Programmed cell death
Internally controlled “suicide” program
Cells are removed with minimal disruption
of the surrounding tissue

4. PathologicalPhysiological
Potentially harmful cells
Outlived their usefulness
Maintain a steady number
Damaged
beyond repair

5. EMBRYOGENESIS

6. Growth factor deprivation

7. Cell loss in proliferating populations
•Eliminate self reactive lymphocytes
•Cell death by induced cytotoxic T lymphocytes
(defense against virus / tumorogenisis)

8. Genetically altered beyond repair
DNA damage (Direct or Free radical)
Radiation
Cytotoxic drugs
Extreme tempetature
Hypoxia

9. Accumulation of misfolded proteins
Arise because of mutations in the genes coding
these proteins or extrensic factors like free
radicals
Cell injury in infections
Atropy in parynchyma after duct obstruction

11. G1 S G2 M
p53
Repair Attempted
NO
YES

12. Initiation
Progression
Termination

13. Cytochrome C
antagonists of
endogenous
cytosolic
inhibitors of
apoptosis
Mitochondria has several proteins capable of
inducing apoptosis
Bcl 2 and bcl XL
antiapoptotic
BAX and BAK
( Pro apoptotic)

14. Dimerize and
insert into
membrane of
mitochondria
Permiability transition
pore Channel activated
(voltage gated channel)
Cytochrome C
CASPASE
ACTIVATION

15. Surface molecules trigger apoptosis
Members of TNF family
Ex: Type 1 TNF and Fas (CD95)
Normally present in cytoplasmic region
On Activation move to surface

16. FasLigand
membrane
protein on
activated T
lymphocytes
activate caspase-8 activating caspase cascade by activating
member of Bcl-2 family called Bid
Bind to Fas

17. CASPASE
Mitochondrial pathway Death receptor pathway
Both pathways are not mutually exclusive with p53 causing cross over to
death receptor pathway.
Cystene protease that cleave proteins after aspartic residues
Cell Death by Apoptosis
Activation of caspase is the fundamental event of apoptosis

18. Caspase-1 (ICE)
Caspase-2 (ICH-1, Nedd-2)
Caspase-3 (CPP32, Apopain, Yama)
Caspase-4 (ICH-2, TX, ICEre
ıı
)
Caspase-5 (ICErel
ııı
, TY)
Caspase-6 (Mch2)
Caspase-7 (ICE-LAP3, Mch3, CMH-1)
Caspase-8 (FLICE, Mch5, MACH)
Caspace-9 (Mch6, ICE-LAP6)
Caspase-10 (Mch4)

19. Caspase activation
a.Proteolytic cleavage
e.g. pro-caspase 3
b.Induced proximity
e.g. pro-caspase 8
c.Oligomerization,
e.g. cyt c, Apaf-1 &
caspase 9

20. Inflammatory Caspases: -1, -4, and -5

Initiator Caspases: -2, -8, -9, and -10
Long N-terminal domain
Interact with effector caspases
Effector Caspases: -3, -6, and -7
Little to no N-terminal domain
Initiate cell death

22. But become avid targets to phagocytosis by flip
out of phosphotidyl serine on the inner leaflet of
plasma membrane
These are recognized by macrophages
Also secrete soluble factors that recruit
phagocytes
Express adhesive glycoproteins recognized by
phagocytes and macrophages

24. CASPASE
.
Cleavage of nuclear LAMINS is involved in chromatin condensation
and nuclear shrinkage.
Cleavage of the inhibitor of the DNase CAD (caspase-activated
deoxyribonuclease), ICAD causes the release of the endonuclease,
which fragment DNA.
Cleavage of cytoskeletal proteins such as actin, plectin, Rho
kinase 1 (ROCK1) and gelsolin leads to cell fragmentation, blebbing
and the formation of apoptotic bodies.

26. Constant number of cells in an organism.
Cell death = Cell proliferation
Cell
Death
Growth
Survival
Proliferation

27. What happens when things become imbalanced?
Cell
Death
Growth
Survival
Proliferation

28. Resistance of tumor cells to apoptosis is an
essential feature of cancer development
Loss of apoptosis can promote tumor
Initiation
Progression
Treatment resistance

29. p53 induce the expression of proteins involved in
the mitochondrial pathway — BAX, NOXA, PUMA and
P53aip1
death receptor pathway — CD95, TRAIL-R1 and TRAIL-
R2.
G1 S G2 M
p53
MalignancyMutation

30. overexpression of anti-apoptotic genes
follicular B-cell lymphoma t(14;18), couples BCL2
gene to immunoglobulin heavy chain locus, leading
to enhanced BCL2 expression.
EBV and HHV8 encode proteins that are
homologues of BCL2.
BHRF1 from EBV and KSbcl-2 (vBcl-2) from HHV8 —
have an anti-apoptotic function and enhance
survival of the infected cells.

31. Adenovirus induce cells to bypass apoptosis
and replicate by E1B oncoprotein inducing cells
to enter S phase
Cytokines like IL6 can inhibit apoptosis

32. soluble receptors that act as decoys for death
ligands.
soluble CD95 (sCD95) and decoy receptor 3 (DcR3)
shown to competitively inhibit CD95 signaling.
sCD95 is expressed in various malignancies, and
elevated levels can be found in the sera of cancer
patients.

33. associated with poor prognosis in melanoma
patients
It is genetically amplified in several lung and
colon carcinomas
overexpressed in several adenocarcinomas,
glioma cell lines and glioblastomas.

34. the pro-apoptotic BCL2 family member BAX is
mutated.
 Frame shift mutations
loss of expression and function.
Reduced BAX expression is associated with a
poor response rate to chemotherapy and
shorter survival

35. Expression of anti-apoptotic proteins
High levels of FLIP which interferes with
apoptosis induction at the level of the death
receptors
Human melanomas
murine B-cell lymphoma
FLIP:caspase-8 ratio cause resistance to
CD95-mediated apoptosis
EBV-positive Burkitt's lymphoma
Viral analogues of FLIP, called viral FLIPs
(v-FLIPs)

36. Expression of anti-apoptotic proteins
Expression of the IAP-family protein
survivin is highly tumour specific.
Found in most human tumours but not
in normal adult tissues.
In neuroblastoma expression correlates
with a more aggressive and
unfavourable disease.

37. Bcl2 shows discrepancies in effect on tumor
management
Studies have shown a correlation between
high levels of BCL2 expression and the
severity of malignancy of human tumours.
a high level of BCL2 expression is associated
with a poor response to chemotherapy and
seems to be predictive of shorter, disease-
free survival.
While in some cases Bcl-2 has been shown to
cause chemo resistance in other settings it is
shown to improve prognosis.

38. Clinical studies examine single alterations
Cannot exclude extragenic mutations in the
same pathway
Thus almost impossible to determine negative
results
Ex: murine lymphomas harbouring INK4a/ARF
have defective p53 but harbour wild type p53
hence classified as p53 normal
Extracellular survival factors affect cell
death
Cell density
microenvironment

39. Application in Treatment
Chemotherapy, irradiation and other stimuli
can initiate apoptosis through the
mitochondrial (intrinsic) pathway.
Pro-apoptotic BCL2 family proteins are
activated by treatment

40. Disrupted apoptotic cycle can affect sensitivity
of cancer drugs
As multiple drugs affect same mechanism it
causes multidrug resistance
Sufficient doses of almost all anticancer drugs
induce apoptosis by alternate pathway
independent of p53 pathway
Contribution of p53 is dependent on
Agent
Dose
mutational background

41. The best-defined mechanism of therapy-induced
cellular stress induced cancer cell death.
Chemotherapeutic drugs (for example, the
nucleotide analogue 5-FU) induce CD95 by a
transcriptionally regulated, p53-dependent
mechanism.
Leads to upregulation of CD95L
Allows the cells to either commit suicide or kill
neighbouring cells.

42. Targeting in therapy
TNF alpha and Fas are toxic to
Normal cells
Cancer cells
But….
TRAIL ---- tumor necrosis factor related
ligand preferentially attacks tumor cells

43. Most mutations occur upstream
Machinery is retained
Tumor specific alterations in apoptotic
programmes
Ex: Adenoviral gene transfer in ovarian cancer
Ad-DF3-Bax eradicated >99% of these tumors in nude
mice
Ex2: Taxanes known to phosphorylate and inactivate
bcl2
inactivating NF-kb enhances chemo induced cell death
Restoring of pro-apoptotic p53
Activating death ligands hence p53 independent
death

44. A Few Add Ons
Apoptosis limited role in solid tumors
Side effects of cancer drugs are because
along with cancer cells they also target
normal cells like intestine and bone marrow

45. Deregulated proliferation alone is not
sufficient for tumour formation
Overexpression of growth-promoting
oncogenes — such as c-MYC, E1A or E2F1 —
sensitizes cells to apoptosis.

46. Cell proliferation Causes apoptosis
IN MALIGNANCY

48. BCL2 family members can be divided into anti-BCL2 family members can be divided into anti-
apoptotic (BCL2, BCL-Xapoptotic (BCL2, BCL-X
LL
, BCL-w, MCL1, A1/BFL1, , BCL-w, MCL1, A1/BFL1,
BOO/DIVA, NR-13) and pro-apoptotic proteins BOO/DIVA, NR-13) and pro-apoptotic proteins
(BAX, BAK, BOK/MTD, BCL-X(BAX, BAK, BOK/MTD, BCL-X
SS
, BID, BAD, , BID, BAD,
BIK/NBK, BLK, HRK/DP5, BIM/BOD, NIP3, NIX, BIK/NBK, BLK, HRK/DP5, BIM/BOD, NIP3, NIX,
NOXA, PUMA, BMF). Most anti-apoptotic members NOXA, PUMA, BMF). Most anti-apoptotic members
contain the BCL2 homology (BH) domains 1, 2 contain the BCL2 homology (BH) domains 1, 2
and 4, whereas the BH3 domain seems to be and 4, whereas the BH3 domain seems to be
crucial for apoptosis induction. The pro-apoptotic crucial for apoptosis induction. The pro-apoptotic
members can be subdivided into the BAX members can be subdivided into the BAX
subfamily (BAX, BAK, BOK) and the BH3-only subfamily (BAX, BAK, BOK) and the BH3-only
proteins (for example, BID, BAD and BIM).proteins (for example, BID, BAD and BIM).