apotosis part 2

5. Initiators of
Apoptosis

6. Caspases
Cysteine-dependent aspartate specific proteases
Have a cysteine at the active site
Cleave target just after aspartic acid residues
Substrate specificity is determined by the 4 residues upstream of
cleavage site( towards N-terminal)
Exist in cytosol as single chain proenzymes (procaspases) which
are activated when cleaved by other caspases
Once activated, cleave other caspases – results in proteolytic
cascade
Also cleave key proteins in the cell, causing the characteristic
morphology and biochemistry of apoptosis.

7. 3 Types of Caspases
• Inflammatory Caspases: -1, -4, and -5
• Initiator Caspases: -2, -8, -9, -10& -12
– Long N-terminal domain
– Interact with effector caspases
• Effector Caspases: -3, -6, and -7
– Little to no N-terminal domain
– Initiate cell death
14 CASPASES identified in humans.

11. Antiapoptotic
Proapoptotic
Bcl-2 family members
A very large family with 30 members identified and belongs to both:
Bid
Bim
Bik
Bad
Bmf
Hrk
Noxa
Puma
Blk
BNIP3
Spike
BH1, BH2,BH
3,BH4
BH
3
BH1, BH
2,BH3

12. THE BCL-2 FAMILY
Raf-1
calcineurin
Ligand
domain
N
Pore
formation
Membrane
anchor
phosphorylation Receptor domain
C

13. Regulatory interactions between Bcl-2 family members

15. Youle and Strasser (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nature
Reviews Molecular Cell Biology, 9, 47-59
BH3 only protein binding specificity for BCL-2 homologues
BIM and PUMA bind to all BCL-2 family members tested; by contrast
NOXA only binds to A1 and MCL1.
These binding specificities recapitulate the ability of these proteins to
activate apoptosis e.g. BIM et al can induce apoptosis alone whereas
a combination of NOXA and BAD is required.

18. IAPs (Inhibitors of Apoptosis)
These proteins act inhibiting caspase activity in 2 different ways:
• Direct binding inhibiting the proteolytic activity of caspases
• Marking caspases for ubiquitination and so degradation
Inhibited by SMAC/DIABLO.
Salvesen and Duckett Nat rev mol cell biol (2002)

27. Kinchen and
Ravichandran (2007)
Fig1
Stages of
engulfment of
apoptotic cells
can be divided
into 4 stages
Binding
Recognition
Phagocytosis
Internalization

28. Lauber et al.(2004) Fig 2 Lack of “don’t eat me” signals on the
surface of apoptotic cells

30. Anterior chamber of the eye and testes fail to elicit an immune response because
these cells produce lots of FasL, so kill immune cells when they enter these
sites.
Possible therapy by inducing production of FasL in other tissues – lowering
need for immune rejection drugs
ORGAN TRANSPLANTS

31. (HIV deactivates Bcl-2)
(

39. 1. p53 mediates apoptosis in response to DNA damage, oncogene
expression (adenovirus E1A, myc etc.), or withdrawal of growth
factors
2. Overexpression wild-type of p53 leads to apoptosis
3. p53 induces apoptosis through transcriptional activation of
proapoptotic genes, such as Puma, Noxa, p53AIP1, Bax, Apaf-1 etc.
4. DNA-damage leads to mitochondrial translocation of p53.
4.p53 binds to Bcl-2 family protein Bcl-xL to influence cytochrome c
release.
p53 in apoptosis

40. p53 binds to Bcl-xL and releases Bax
.
Bax is sequestered by
Bcl-xL and inactive
Bax is released by p53
from Bcl-xL and forms
oligomers, leading to
apoptosis
. p53 can release both proapoptotic multidomain proteins
and BH3-only proteins

41. p53 and Puma in
apoptosis
Ref: K.
Vousden, Science,
2005

42. p53 in DNA repair
and apoptosis
Ref: Bensaad &
Vousden, Nature
Med, 2005
ROS: reactive oxygen species
G1- S

43. Role of P53 in DNA damage induced Apoptosis
• DNA damage activate
ATM & Chk2 protein
kinases
• Phosphorylation of P53
• Activate Cdk inhibitor
P21, which inhibits
Cdk2/cyclin E
complexes, halting cell
cycle progression in G1
NOXA

44. The p53-MDM2 feedback loop
1. MDM2 binds to p53 N-terminal transactivation
domain and inhibits p53-dependent transcription.
2. MDM2 is a transcription target of p53.
3. MDM2 is an E3 ubiquitin ligase of p53, thus
targeting p53 for proteolytic degradation.
4. MDM2 knockout is lethal for mouse embryonic
development, but simultaneous deletion of p53
and MDM2 genes rescues MDM2-KO.

45. The p53 pathways

46. Ref: Nature 408, 307 - 310 (2000)
Viral oncogenes and the p53 network

47. Genetic Control of "Genetically
Programmed Cell Death"
Cellular Location of
Protein Product
Mitochondrial Membrane
Nuclear Envelope
Endoplasmic Reticulum
Nucleus
Nucleus
Cell Membrane
Effect on Apoptosis
Blocks
Stimulates
Wild-type Stimulates
Mutant Blocks
Stimulates
ApoGenes
bcl-2
myc
p53
p53*
APO-1/
FAS
Apoptosis
Gene

48. Tumor-derived mutations affecting
apoptosis
Protein Role in apoptosis Ref
ATM Mutated in ataxia-talangiectasia syndrome. Senses DNA
double strand breaks and stabilizes p53. Deficiencies
increase risk of developing haematological malignancies
and breast cancer
Khanna and
Jackson, 2001
Bax (p53 target
gene)
Mutated or decreased expression in some tumors.
Mediates mitochondrial membrane damage. Sufficient
but not necessary for drug-induced apoptosis.
Rampino et al.,
1997
Bak Mutated or decreased expression in some tumors.
Mediates mitochondrial membrane damage. Sufficient
but not necessary for drug-induced apoptosis.
Kondo et al., 2000
PTEN (p53
target gene)
Mutated or altered expression in cancers. Regulates Akt
activation and subsequent phosphorylation of Bad. Loss
of PTEN results in resistance to many apoptotic stimuli.
Di Cristofano and
Pandolfi, 2000
Ref: Cell, 2002, 108:153-164

49. Tumor-derived mutations affecting
apoptosis
Protein Role in apoptosis Ref
Apaf-1 (p53
target gene)
Mutated and transcriptionally silenced in melanoma
and leukemia cell lines. Necessary for activation of
caspase-9 following cytochrome c release. Apaf-1-/-
cells are chemoresistant.
Soengas et al., 2001
CD-95/Fas Mutated and down-regulated in lymphoid and solid
tumors. Initiates the extrinsic apoptotic pathway. Loss
of function is associated with resistance to drug-
induced cell death.
Muschen et al., 2000
TRAIL-
R1/R2
Mutated in metastatic breast cancers. Initiate the
extrinsic apoptotic pathway. Mutations lead to
suppression of death receptor-mediated apoptosis.
Shin et al., 2001
Caspase-8 Gene silenced in neuroblastomas. Activates both
extrinsic and intrinsic apoptotic pathways. Silencing
results in resistance to drug-induced apoptosis.
Teitz et al., 2000
Ref: Cell, 2002, 108:153-164

50. Tumor-derived mutations affecting
apoptosis
Protein Role in apoptosis Ref
Bcl2 Frequently overexpressed in many tumors. Antagonises
Bax and/or Bak and inhibits mitochondrial membrane
disruption. Inhibits drug-induced apoptosis.
Reed, 1999
MDM2 Overexpressed in some tumors. Negative regulator of
p53. Inhibits drug-induced p53 activation.
Sherr and Weber,
2000
IAPs Frequently overexpressed in cancer. Down regulation
of XIAP induces apoptosis in chemoresistant tumors.
Deveraux and Reed,
1999
NF B Deregulated activity in many cancers. Transcriptionally
activates expression of anti-apoptotic members of the
Bcl-2 and IAP families. Can inhibit both the extrinsic
and intrinsic death pathways and induce drug-
resistance.
Baldwin, 2001
Ref: Cell, 2002, 108:153-164

51. Tumor-derived mutations affecting
apoptosis
Protein Role in apoptosis Ref
p53 Mutated or altered expression in many cancers.
Initiates the intrinsic apoptotic pathway. p53-/-
cells are resistant to drug induced apoptosis.
Vogelstein et al., 2000
p19ARF Mutated or altered expression in many cancers.
Blocks MDM2 inhibition of p53. Enhances drug-
induced apoptosis by p53.
Sherr and Weber, 2000
Rb Mutated in some cancers, and functionally
disrupted in many cancers. Inhibits E2F-
medidated transcription. Loss of Rb function
induces p53-dependent and independent apoptosis.
Harbour and Dean,
2000
Chk2 Mutated in Li-Fraumeni syndrome. Senses DNA
double strand breaks and phosphorylates and
stabilizes p53.
Khanna and Jackson,
2001
Ref: Cell, 2002, 108:153-164

52. Tumor-derived mutations affecting
apoptosis
Protein Role in apoptosis Ref
Myc Deregulated expression in many cancers. Induces
proliferation in the presence of survival factors, such as
Bcl-2, and apoptosis in the absence of survival factors.
Can sensitise cells to drug-induced apoptosis.
Evan and Vousden,
2001
Akt Frequently amplified in solid tumors. Phosphorylates
Bad. Hyperactivation induces resistance to a range of
apoptotic stimuli including drugs.
Datta et al., 1999
PI3K Overexpressed or deregulated in some cancers.
Responsible for activation of Akt and downstream
phosphorylation of Bad. Inhibition of PI3K enhances
chemotherapeutic drug-induced apoptosis.
Roymans and
Slegers, 2001
Ras Mutated or deregulated in many cancers. Activates PI3K
and downstream pathways. Induces proliferation and
inhibits c-myc and drug-induced apoptosis.
el-Deiry, 1997
Ref: Cell, 2002, 108:153-164

53. Tumor-derived mutations affecting
apoptosis
Protein Role in apoptosis Ref
FLIP Overexpressed in some cancers. Prevents activation of
caspase-8 and apoptosis induced by some
chemotherapeutic drugs.
Tepper and Seldin,
1999
Ref: Cell, 2002, 108:153-164

54. Tumor advantages following p53
mutations
1. Cell cycle - mutant cells are able to progress
through the cell cycle and divide, passing on
mutations.
2. Apoptosis - these cells ignore signals to commit
cell suicide.
3. Genetic instability - continued division without
checkpoints leads to chromosomal
aberrations, incorrect rejoining of chromosomes
activation of oncogenes and inactivation of tumor
suppressor proteins

55. Therapeutic applications of
regulating apoptosis
Promote apoptosis in
cancer cells:
Lymphoma/leukemia
Oral cancer
Brain tumors
Prostate
Colon
etc.
Prevent apoptosis in
certain disorders and
degenerative diseases:
AIDS
Ischemia
Alzheimer's/Parkinson's
etc.

56. Apoptosis in the treatment of cancer
An important goal of cancer drug development should be to
facilitate apoptosis in neoplastic cells. Drugs that restore apoptosis
might selectively kill cancer cells that have triggered a death signal
and have become dependent on the deregulation of apoptosis
pathways.
Strategies already used:
• Administration of death ligand
• Bcl-2 family inhibitors
• XIAP inhibitors
Fesik Nat Rev Cancer (2005)

58. Ribozyme Inhibition of Bcl-2 Expression
• Apoptosis removes damaged cells from the body. The
bcl-2 gene prevents this.
• The role of bcl-2 in oral cancer and glioblastoma is
unexplored.
• We constructed a hammerhead ribozyme that would
digest the bcl-2 mRNA message and delivered it to oral
cancer and glioblastoma cells with an adenovirus
vector.

59. Ref: Nature, 412, 865 - 866 (2001)
AAV kills cancer cells

62. 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)

65. „Don't think of death as an ending. Think of it as a really effective way of
cutting down your expenses.” Woody Allen