The Biology of Cancer
Chapter 9:
p53 and Apoptosis: Master
Guardian and Executioner
Why p53?
• Need to eliminate malfunctioning cells
• Must have a decision maker (p53)
• If too much damage, initiates programmed
cell suicide (Apoptosis)
• P53 is dangerous for the cancer cell because
its job is to kill cancer cell
Discovery of p53
• Originally identified as an oncogene in
association with Large T antigen in SV40
• Over-expressed in murine tumors
• Forms found in tumors are mutant, have to
amino acid changes in functional domains
• Does not conform to Knudson’s “two hit
hypothesis” model
How can a mutant form foster tumor
growth?
• Yeast: Mutant alleles of gene can interfere
with wild-type gene function
• “Dominant-interfering” or “DominantNegative”
• Wild-type P53 found to be a homo-tetramer
(made from 4 identical units)
• Single mutation produce normal and error
copies of each unit in equal proportion
Figure 9.7a The Biology of Cancer (© Garland Science 2007)
Out of 16 tetramers, only one will function normally
Figure 9.7b The Biology of Cancer (© Garland Science 2007)
What happens when there is a
mutation in p53?
• Mutant form makes tetramer but it cannot
function normally
• P53 efficiency reduced to 1/16 th of normal
• Advantage for tumors
• Many tumors also have LOH at mutation
locus for p53
• They cannot tolerate even 1/16 efficiency of
p53
Figure 9.4 The Biology of Cancer (© Garland Science 2007)
Variation in type of mutation seen in different genes in cancer
Figure 9.6a The Biology of Cancer (© Garland Science 2007)
Most p53 mutations are in the DNA binding domain
Figure 9.6b The Biology of Cancer (© Garland Science 2007)
P53 has a short half-life
• Cycloheximide blocks protein synthesis
• When added to cells with wild-type p53
in-vitro, amount of p53 protein decayed
with ½ life of 20 minutes
• p53 has a “futile” cycle
• Useful way to create a “fast switch” for a
critical protein. Turns on immediately when
its degradation is inhibited.
What activates p53?
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X-rays, UV radiation damage
DNA damaging chemo agents
Inhibitors of DNA synthesis
Microtubule disruption
Many carcinogens, drugs
Hypoxia
High Nitrous Oxide concentrations
More activation signals
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Oncogene upregulation
Changes in methylation of chromosomes
Acidified growth medium
Reduction in ribonucleotides
Blocks in RNA/DNA synthesis
• P53 causes “growth arrest” if it senses damage
• Initiates a “Repair” program
• If damage persists or repair fails, initiates cell suicide
Figure 9.8 The Biology of Cancer (© Garland Science 2007)
How p53 stops cell cycle
• X rays upregulate p53 which induces p21Cip1
• p21 binds to Cyclin D, CDK4/6
• X-rays do not induce
p21 when p53 mutated
• Direct correlation seen in
p53 levels and apoptotic
rate for different amounts
of X-ray
Figure 9.9 The Biology of Cancer (© Garland Science 2007)
p53/MDM2 “futile” cycle
• P53 protein is transcription factor for MDM2
• MDM2 ubiquitylates p53 for degradation in
proteasome
Figure 9.11 The Biology of Cancer (© Garland Science 2007)
• P53 protein recognizes a specific DNA sequence:
Pu-Pu-Pu-C-A/t-T/a-G-Py-Py-Py repeated twice with a gap of 0-13
• MDM2 binds p53 protein in a small N-terminus region
• Grey area allows it to form a tetramer
• Red: NLS or Nuclear Localization Signals + amino acid for DNA binding
• “Proline rich” region contributes its apoptotic activity
Figure 9.12 The Biology of Cancer (© Garland Science 2007)
p53 activation mechanism
• Double stranded DNA breaks (X-rays)
– ATM ATR Kinase  phosphorylation of p53
to stabilize it
• DNA damaging agents
– ATM CKII phosphorylate p53
• Aberrent Growth signals
– e.g. (pRb-E2F) disregulation: Mechanism ?
• Hypoxia: Mechanisms?
• P53 stabilized by
phosphorylation
• Phosphorylation done by
kinases like ATM, Chk1,
Chk2 which are activated by
DNA damage
• They alter the p53 domain
recognized by MDM2
• ATM can also phosphorylate
MDM2 to deactivate it
ATM  p53
ATM --| MDM2
MDM2 --| p53
Figure 9.13 The Biology of Cancer (© Garland Science 2007)
MDM2 is a “funny” oncogene
• Acts by “antagonizing” a tumor suppressor
• Prevents entry into cell cycle arrest, senescence,
cell suicide
• MDM2 is under control of other proteins:
– E.g. Ras Raf  MapK  ETS/AP-1 which through
FOS + Jun transcription factors can increase MDM2
levels
• SNP 309 in MDM2 intron is cancer promoting
ARF
P14
• ARF = Alternate Reading Frame
• Transcriptional promotor 13 kb upstream of
gene for p16INK4A deletes exon 1 and an
alternate reading frame in Exon 2
• Result = ARF mRNA
Figure 9.14 The Biology of Cancer (© Garland Science 2007)
ARF is a tumor suppressor
• Shuts down MDM2 by sequestering it in
nucleolus
• ARF + p53 monitor intracellular signalling
and cell cycle
– pRb --| E2F  ARF --| MDM2 --| p53  death
• p14ARF has an E2F recognition sequence
• Elimination of ARF removes check on E2F
levels
Figure 9.15b The Biology of Cancer (© Garland Science 2007)
Table 9.2 The Biology of Cancer (© Garland Science 2007)
P53 is not involved in “routine” cell death e.g. in morphogenesis
Mouse hand morphogenesis. Black
dots are cells undergoing apoptosis
Figure 9.19 The Biology of Cancer (© Garland Science 2007)
Li-Fraumini Syndrome
• Predisposition to Glioblastomas,
Leukemias, Breast, Lung, Pancreatic tumors
• Mandelian inheritance of trait
• Traced to scattered mutations in p53 gene
on Chr17-p13
Figure 9.20 The Biology of Cancer (© Garland Science 2007)
The bcl-2 Oncogene
• B-Cell Lymphoma Gene seen to be upregulated in tumors
• Mutation is reciprocal translocaton in Chr14/Chr18
• Mutant bcl-2 inserted in mouse germline and expressed in
lymphocyte precursor has no effect on survival
• Myc upregulation + bcl-2 mutation killed mice in < 2
months
• Myc upregulation is also potent killer but not as
potent as Myc + bcl-2
Figure 9.22b The Biology of Cancer (© Garland Science 2007)
Mechanism of action of bcl-2
• Mutant bcl-2 prolongs survival of
lymphocytes in-vitro
• In vivo, prolongs lymphocyte life but cells
are not proliferating  no tumors induced
• Bcl-2 seems to act in the “opposite way”
from p53, causing a halt in “death” program
Figure 9.27c The Biology of Cancer (© Garland Science 2007)
Apoptosis and bcl-2
• bcl-2 is anti-apoptotic
• Found localized in outer membrane of
mtDNA !
• Apoptosis is triggered by depolarization of
outer membrane of mtDNA and release of
Cytochrome C
• Cytochrome C associates with other
proteins to initiate cell death
Apoptosis =
“Programmed Cell Death”
Figure 9.29 The Biology of Cancer (© Garland Science 2007)
Two apoptotic pathways
• P53 induced – driving genes
like Bax which open
mtDNA channels
• Extracellular, initiated by
signals from
transmembrane
“death receptors”
There is a third pathway
• T-cell and NK cell mediated
• Immune system
• Killer cells attach a protease (like a bomb)
to cell outer membrane. When internalized
it cleaves and activates procaspase 3,8,9
 Cell death
Convergence of Intrinsic and Extrinsic Apoptotic pathways
Figure 9.32 The Biology of Cancer (© Garland Science 2007)
P53 can also use the extrinsic pathway by
• inducing a gene for the Fas “death” receptor
• inducing IGFBP-3 which binds to extracellular IGF-1,2 ligands
to reduce effect of external anti-apoptotic signals
Figure 9.33 The Biology of Cancer (© Garland Science 2007)
Anti-Apoptotic Strategies of Cancer Cells
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Point mutations in p53 to disable pathways
Deletion or promotor methylation of ARF
Overexpression of MDM2
Mis-localization of p53 (sequestering in cytoplasm)
Hijacking of apoptotic pathway (Melanoma)
Inactivating Bax (Colon Cancer)
Disregulating bcl-2 (Follicular Lymphoma)
Hyperactivation of PI3K  Aft  PkB pathway
Activation of NF-kB pathway
Anti-apoptotic Pathway:
Figure 9.34 The Biology of Cancer (© Garland Science 2007)
Table 9.5 The Biology of Cancer (© Garland Science 2007)
Different post transcriptional modifications of p53 protein
Figure 9.35 The Biology of Cancer (© Garland Science 2007)
Autophagy or Cell Self-Cannibalism
• A survival mechanism. Is it a mechanism for therapy?
• Beclin-1 induces autophagy and is down-regulated by tumors
Figure 9.36 The Biology of Cancer (© Garland Science 2007)
The p53 circuit board
Figure 9.37 The Biology of Cancer (© Garland Science 2007)