GMS 6644
Apoptosis
Lecture 5: p53 and Apoptosis
R4-265
Mar 1, 06, 9 – 10:30 pm
Dr. Daiqing Liao B1-016
dliao@ufl.edu
Tumor-suppressor genes
Tumor-suppressor genes, function like brakes, keep
cell numbers down, either by inhibiting progress
through the cell cycle and thereby preventing cell
birth, or by promoting programmed cell death (also
called apoptosis). When cellular tumor suppressor
genes are rendered non-functional through
mutation, the cell becomes malignant. Examples are
the gene encoding the retinoblastoma protein (Rb),
inactivated in retinoblastomas, p53, and p16INK4a,
which inhibits cyclin-dependent kinases and is
inactivated in many different tumors.
Oncogenes
Oncogenes stimulate appropriate cell growth under
normal conditions, as required for the continued
turnover and replenishment of the skin,
gastrointestinal tract and blood, for example. Cells
with mutant oncogenes continue to grow (or refuse
to die) even when they are receiving no growth
signals. Examples are Ras, activated in pancreatic
and colon cancers, and Bcl-2, activated in lymphoid
tumours. Amplification of oncogenes (more than
their normal gene copy number) is also found in
cancer: MDM2 is amplified in liposarcomas.
Roles of p53 in apoptosis
1. p53 induces apoptosis through transcriptional
activation of proapoptotic genes, such as Puma,
Noxa, p53AIP1, Bax, Apaf-1 etc.
2. It can also directly induce apoptosis by
localizing to mitochondria via interaction with
Bcl-2 family protein Bcl-xL and facilitating Bax
oligomerization
Reading: Vousden and Lu: Nature Reviews
Cancer, 2002, 2:594-604.
p53 and of apoptosis
Ref: Mol. Cell, 2003, 11(3):552-4
p53 and apoptosis
Ref: Cell, 2002, 108:153-164
Tumor Suppressor p53
• First identified as a protein associated with viral
oncogenes
• Mutated/inactivated in a majority of human
cancers
• Integrates numerous signals that control cell life
and death
• A common denominator in human cancer
• Understanding functions and regulation of p53
is of great importance in cancer biology and
cancer therapy
The p53 pathways
The p53 network
p53 is a sequence-specific DNA
binding protein
1. p53 central core-domain interacts directly with DNA
2. p53 binding sites consist of four copies of the pentamer
consensus sequence PuPuPuC(A/T). The pentamers are
oriented in alternating directions. A short stretch of
sequence up to 13 bp may be inserted between the pairs
of pentamers. The p53 target genes in the human genome
usually carry the consensus sequence.
3. Amino acid residues in the core-domain that are critical
for DNA-binding are among the “hot-spots” of tumorderived p53 mutations, attesting to the importance of
DNA-binding for p53’s tumor suppression function.
Structure of p53 core-domain
Ref: Science, 265:346-355, 1994
Structure of the p53 OD
Science 1995, 67:1498-1502.
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, thus
confirming the in vivo genetic interaction of
these two proteins.
Structure of MDM2-p53 complex
Science 1996 274:948-953
p53 and apoptosis
Apaf-1
p53AIP1
p53 in apoptosis
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 can induce the expression of proapoptotic
genes, such as Bax (ref Cell, 80:293) and
p53AIP1 (ref: Cell, 102:849)
p53 in apoptosis
4. p53 can also repress transcription of certain
genes, and it has been proposed that the
repression function may also be required for
apoptosis (ref: Genes Dev 13:2490-501)
5. In vivo, p53 transactivation mutant is defective
in inducing apoptosis, at least for some cell types
(ref: EMBO J. 19:4967-4975)
Physiological relevance of p53induced apoptosis
• Suppress oncogene-induced transformation
• Inhibit tumor growth and progression
• Remove cells with severe DNA damage
• Effectiveness of cancer chemotheraphy correlates
with the ability to induce p53-dependent
apoptotic response
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 druginduced apoptosis by p53.
Sherr and Weber, 2000
Rb
Mutated in some cancers, and functionally
disrupted in many cancers. Inhibits E2Fmedidated 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
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
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
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
gene)
target gene)
Ref: Cell, 2002, 108:153-164
Tumor-derived mutations affecting
apoptosis
Protein
Role in apoptosis
Ref
Apaf-1 (p53
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 druginduced cell death.
Muschen et al., 2000
TRAILR1/R2
Caspase-8
Mutated in metastatic breast cancers. Initiate the
extrinsic apoptotic pathway. Mutations lead to
suppression of death receptor-mediated apoptosis.
Shin et al., 2001
Gene silenced in neuroblastomas. Activates both
extrinsic and intrinsic apoptotic pathways. Silencing
results in resistance to drug-induced apoptosis.
Teitz et al., 2000
target gene)
Ref: Cell, 2002, 108:153-164
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
NFkB
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 drugresistance.
Baldwin, 2001
Ref: Cell, 2002, 108:153-164
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
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
p53 directly mediates mitochondrial
mechanism of apoptosis
1. Early studies indicate that transactivation-defective mutants
of p53 are capable of inducing apoptosis, implying a
transcription-independent role for p53 in apoptosis.
2. DNA-damage leads to mitochondrial translocation of p53.
3. p53 binds to Bcl-2 family protein Bcl-xL to influence
cytochrome c release.
4. p53 directly activates the proapoptotic Bcl-2 protein Bax in
the absence of other proteins to permeabilize mitochondria
and engage the apoptotic program.
5. p53 can release both proapoptotic multidomain proteins and
BH3-only proteins [Proapoptotic Bcl-2 family proteins that
share only the third Bcl-2 homology domain (BH3)] that are
sequestered by Bcl-xL.
Ref: Mol. Cell, 2003, 11:577-90; Science, 2004, 303:1010-4.
p53 is found in mitochondria following DNA
damage
Ref: Mol. Cell, 2003, 11:577-90
Recombinant p53 causes rapid cytochrome c
release from mitochondria
Ref: Mol. Cell, 2003, 11:577-90
Cooperation of endogenous cytoplasmic p53 with Bax to
induce apoptosis in the absence of p53 nuclear activity
Ref: Science, 2004, 303:1010-4.
How p53 functions in mitochondria to
induce cell death
1. p53 or Bax alone does not permeabilize
membrane, but they together can do so.
2. p53 facilitates Bax oligomerization.
3. p53 binds to Bcl-xL, but not to Bax.
4. p53-Bcl-xL interaction releases Bax.
5. Released Bax forms oligomers in
mitochondrial membrane, leading cytochrome
c release and apoptosis.
6. The proline-rich domain (aa 62-91 in mouse) of
p53 is required for this effect.
Ref: Science, 2004, 303:1010-4.
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
Ref: Science, 2004, 303:1010-4.
p53 and Puma in apoptosis
Ref: K. Vousden,
Science, 2005
Viral oncogenes and the p53 network
Ref: Nature 408, 307 - 310 (2000)
Viral oncogenes inhibit p53 functions
1. p53 was first identified as binding protein of
SV40 large T antigen in 1979, which interacts
with p53 core domain
2. Adenovirus oncoprotein E1B 55-kDa binds to
p53 and inhibits p53 transactivation activity
3. HPV E6 binds to p53 and targets it for ubiquitinmediated degradation.
Ad12 E1B and p53 colocalize in the
cytoplasmic bodies
Ref: Zhao&Liao. J. Virol., 2003,77:13171-13181.
Inhibition of p53-mediated apoptosis by E1B
55-kDa through cytoplasmic sequestration
• Ad12 E1B 55-kDa forms large and
filamentous cytoplasmic body
• p53 localizes to the E1B cytoplasmic body,
and its N-terminal TAD is required for this
localization
• Sequestration of p53 in the cytoplasmic
body correlates with the ability of E1B 55-kDa
to repress p53-dependent apoptosis
Using p53 to kill cancer cells
The p53 protein is a tumor suppressor — it keeps cell numbers
down by stopping cells from multiplying or by promoting
cell death. Loss of p53 occurs in most human cancers, so it
would be useful to be able to restore its function. Several
innovative strategies have been suggested:
• Introduce normal p53 genes into a cancer cell with mutant
p53.
• Introduce a small compound that converts mutant p53
proteins from an abnormal to a normal shape.
• Add a protein that attaches itself to mutant p53 and kills
cells.
• Stimulate the host's immune response to mutant p53
peptides.
Using p53 to kill cancer cells
• Introduce drugs that disrupt the interaction between the
MDM2 or E6 proteins and p53. (MDM2 and E6
negatively regulate p53; they are present at abnormally
high levels in some cancer cells, so 'quench' any normal
p53.)
• Introduce the adeno-associated virus, which mimics
damaged DNA. Cells with mutant p53 cannot activate
the usual p53-dependent 'checkpoint' that is induced by
DNA damage, and eventually die.
• Infect cells with viruses that can replicate only in cells
without normal p53; the viruses kill these cells.
AAV kills cancer cells
Ref: Nature, 412, 865 - 866 (2001)
p53 in DNA repair and apoptosis
ROS: reactive oxygen species
Ref: Bensaad &
Vousden, Nature
Med, 2005
Summary
1. p53 is a tumor-suppressor protein that induces apoptotic cell
death in response to oncogenic stress. Malignant progression
is dependent on loss of p53 function, either through mutation
in the TP53 gene (which encodes p53) itself or by defects in
the signaling pathways that are upstream or downstream of
p53.
2. Mutations in TP53 occur in about half of all human cancers,
almost always resulting in the expression of a mutant p53
protein that has acquired transforming activity.
3. p53-induced apoptosis depends on the ability of p53 to
activate gene expression.
4. p53 can also directly trigger the apoptotic response, by
interacting with Bcl-2 family protein.
5. The apoptotic and cell-cycle arrest activities of p53 can be
separated, and apoptotic cofactors that play a specific role in
allowing p53-induced death are being identified.
Ref: Nature Reviews Cancer, 2, 594 - 604 (2002)
Summary
6. Phosphorylation of p53 regulates its ability to activate the
expression of apoptotic target genes, and other posttranslational modifications such as acetylation might also
have a role.
7. In tumors that retain wild-type p53, the apoptotic response
might be hindered by defects in the apoptotic cofactors.
These, therefore, represent additional targets for the design
of therapeutics that are aimed at reactivating p53-mediated
apoptosis in cancer cells.
Ref: Nature Reviews Cancer, 2, 594 - 604 (2002)