GMS 6644 Apoptosis

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GMS 6644 Apoptosis

• Lecture 5 p53 and Apoptosis
• R4-265
• Mar 1, 06, 9 1030 pm
• Dr. Daiqing Liao B1-016
• dliao_at_ufl.edu

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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.
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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.
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Roles of p53 in apoptosis

• p53 induces apoptosis through transcriptional
  activation of proapoptotic genes, such as Puma,
  Noxa, p53AIP1, Bax, Apaf-1 etc.
• 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, 2594-604.
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p53 and of apoptosis
Ref Mol. Cell, 2003, 11(3)552-4
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p53 and apoptosis
Ref Cell, 2002, 108153-164
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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

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The p53 pathways
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The p53 network
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p53 is a sequence-specific DNA binding protein

• p53 central core-domain interacts directly with
  DNA
• 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 tumor-derived p53 mutations,
  attesting to the importance of DNA-binding for
  p53s tumor suppression function.

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Structure of p53 core-domain
Ref Science, 265346-355, 1994
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Structure of the p53 OD
Science 1995, 671498-1502.
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The p53-MDM2 feedback loop

• MDM2 binds to p53 N-terminal transactivation
  domain and inhibits p53-dependent transcription.
• 2. MDM2 is a transcription target of p53.
• 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.

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Structure of MDM2-p53 complex
Science 1996 274948-953
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p53 and apoptosis
p53AIP1
Apaf-1
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p53 in apoptosis

• 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, 80293) and p53AIP1
  (ref Cell, 102849)

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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 132490-501) 5. In
vivo, p53 transactivation mutant is defective in
inducing apoptosis, at least for some cell types
(ref EMBO J. 194967-4975)
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Physiological relevance of p53-induced 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
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Tumor-derived mutations affecting apoptosis
Ref Cell, 2002, 108153-164
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Tumor-derived mutations affecting apoptosis
Ref Cell, 2002, 108153-164
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Tumor-derived mutations affecting apoptosis
Ref Cell, 2002, 108153-164
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Tumor-derived mutations affecting apoptosis
Ref Cell, 2002, 108153-164
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Tumor-derived mutations affecting apoptosis
Ref Cell, 2002, 108153-164
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Tumor-derived mutations affecting apoptosis
Ref Cell, 2002, 108153-164
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p53 directly mediates mitochondrial mechanism of
apoptosis

• Early studies indicate that transactivation-defect
  ive mutants of p53 are capable of inducing
  apoptosis, implying a transcription-independent
  role for p53 in apoptosis.
• DNA-damage leads to mitochondrial translocation
  of p53.
• p53 binds to Bcl-2 family protein Bcl-xL to
  influence cytochrome c release.
• p53 directly activates the proapoptotic Bcl-2
  protein Bax in the absence of other proteins to
  permeabilize mitochondria and engage the
  apoptotic program.
• 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, 11577-90 Science, 2004,
3031010-4.
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p53 is found in mitochondria following DNA damage
Ref Mol. Cell, 2003, 11577-90
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Recombinant p53 causes rapid cytochrome c release
from mitochondria
Ref Mol. Cell, 2003, 11577-90
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Cooperation of endogenous cytoplasmic p53 with
Bax to induce apoptosis in the absence of p53
nuclear activity
Ref Science, 2004, 3031010-4.
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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, 3031010-4.
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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, 3031010-4.
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p53 and Puma in apoptosis
Ref K. Vousden, Science, 2005
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Viral oncogenes and the p53 network
Ref Nature 408, 307 - 310 (2000)
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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 ubiquitin-mediated degradation.
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Ad12 E1B and p53 colocalize in the cytoplasmic
bodies
Ref ZhaoLiao. J. Virol., 2003,7713171-13181.
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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
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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.
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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.
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AAV kills cancer cells
Ref Nature, 412, 865 - 866 (2001)
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p53 in DNA repair and apoptosis
Ref Bensaad Vousden, Nature Med, 2005
ROS reactive oxygen species
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Summary

• 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.
• 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.
• p53-induced apoptosis depends on the ability of
  p53 to activate gene expression.
• p53 can also directly trigger the apoptotic
  response, by interacting with Bcl-2 family
  protein.
• 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)
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Summary

• Phosphorylation of p53 regulates its ability to
  activate the expression of apoptotic target
  genes, and other post-translational modifications
  such as acetylation might also have a role.
• 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)