Transcript p53 and Control of the Cell Cycle Learning Objectives 1. Conclude the role of p53 in preserving genome stability (Bloom’s Cognition Level.

p53 and Control of the Cell Cycle
Learning Objectives
1. Conclude the role of p53 in preserving genome
stability (Bloom’s Cognition Level 2, Understand)
2. Relate the functions of p53, MDM2, and Arf to
triggering apoptosis and cell cycle arrest (Bloom’s
Cognition Level 3, Apply)
3. Propose a mechanism for a cell’s response to a
situation in terms of a familiar story (Bloom’s
Cognition Level 6, Create)
A Little About p53…
- Acts as a tumor suppressor gene
- 2 Main Functions:
- halts growth and division in cell cycle under
aberrant conditions
- induces apoptosis
- Loss of p53 function leading cause in 30-50% of
various types of cancers
Mutant Version of p53
Knudson’s model of tumor suppressor genes: loss of one copy
(haploinsufficiency) should reduce function of the gene by ~50%.
-p53 does not follow this pattern: far more than 50% efficiency
in haploinsufficient cells
Experiment: Mutant p53 cDNA introduced in rat embryo fibroblasts
-Point-mutated p53 allele exerted dominant function: most of
the tumor suppression was lost
How does this make sense?
Mutant Version of p53
• Most mutant p53 alleles carry point mutations in reading frames
that create missense codons instead of nonsense codons
• How does mutant p53 foster tumor formation?
– p53 is a dominant-negative allele
– p53 exists as a homotetramer: assembly of four identical
polypeptide subunits
Structure of p53
Because it acts as a tetramer, one mutant p53 can disrupt three healthy p53
proteins in the tetramer.
Cells heterozygous for p53 can still make perfectly functional homotetramers,
but only 1/16 of the possible combinations is healthy.
In human tumor cells mutant at p53 locus, p53 locus found to have undergone
loss of heterozygosity (LOH), in which wild type allele is discarded, yielding a cell
with two mutant alleles.
p53 Protein Molecules Have Short
Lifetimes
• Nuclear localization suggests that p53 protein functions as
a transcription factor (TF)
• 3 mechanisms regulate its activity
– Level of TF in nucleus modulated: can degrade it in proteasomes
– Level of TF in nucleus held constant, but intrinsic activity
regulated by some covalent modifications (phosphorylation,
acetylation, methylation)
– Level of collaborating TFs modulated
• Treat cells with cycloheximide (protein synthesis
inhibitor)- p53 disappeared with half life of 20 minutes
• Conclude: p53 is a highly unstable protein
Variety of Signals Cause p53 Induction
• Receives signals from a diverse array of surveillance
systems
– Increased expression of TF called E2F1, de-methylation of
chromosomal DNA, exposure to nitrous oxide, blockage of RNA or
DNA synthesis can increase p53 levels
• Genotoxic (DNA damaging) agents and physiological
signals increase p53 levels to act in cytostatic fashion
(growth arrest or apoptosis)
Contd..
• Hypoxia (lowered oxygen tension), genomic
damage, imbalances in signaling pathways
governing cell proliferation are experienced by
cancer cells
• In response, functional p53 alarm system
triggered to activate p53’s function
• p53 acts as the “guardian of the genome”
Mdm2 and ARF Battle
Over the Fate of p53
• Mdm 2 recognizes and binds the N-terminal
domain of p53
• Blocks the ability of p53 to act as a
transcription factor
• Promotes p53 export from the nucleus
Mdm2 and ARF Battle
Over the Fate of p53
• Mdm2 targets p53 for degradation
• Adds a ubiquitin moiety
• Upon export to the cytoplasm polyubiquitylation
occurs
• Negative feedback loop
– p53 promotes Mdm2 gene expression
– Mdm2 targets p53 for destruction
Mdm2 and ARF Battle
Over the Fate of p53
• Phosphorylation of Mdm2 plays an important
role
– Multiple sites on Mdm2 can be phosphorylated
– Phosphorylation can activate or inhibit Mdm2
function
• Survival signals like Akt phosphorylate and
activate Mdm2, leading to cell survival
• ATM and ATR phosphorylate and inactivate
Mdm2, at the p53 binding domain of Mdm2.
The inhibition of Mdm2 permits p53 activation
Mdm2 and ARF Battle
Over the Fate of p53
• Is p53 helpless in its defense from Mdm2?
• No! ARF binds to Mdm2
– Prevents it from binding p53
– Sequesters it in nucleolus
• p53 escapes ubiquitylation, can rise to high cellular
levels
Mdm2 and ARF Battle
Over the Fate of p53
• Need balance in the regulation of ARF, p53
and Mdm2
• Over/Under expression of Mdm2 leads to
cellular disruption
– Mouse embryo cells without Mdm2
expression do not proliferate
– Over expression of Mdm2 can lead to excess
proliferation
Mdm2 and ARF Battle
Over the Fate of p53
• Mdm2 promoter critical in expression of
Mdm2
– Ease of transcription factor binding increases
expression of Mdm2
– More in next session….
Activity 1 (5 min)
What characters in the movie Batman:
The Dark Knight best represent p53,
Mdm2, and ARF, and why? (Use
handout to record your answers)
http://www.youtube.com/watch?v=FXMB5KNYgMw&feature=related
Activity 1 – Results
• p53: Batman – Guards the genome, protects
Gotham
• Mdm2: Joker – Inhibits p53, creates chaos by
inactivating Batman
• ARF: Gordon—Binds mdm2, prevents it from
binding p53; helps Batman by jailing the Joker
Why Is Batman p53?
• Guardian of Gotham city (genome)
• Acts to prevent crime (cell cycle arrest), and
when necessary destroy evil (apoptosis)
• Unstable depiction of heroism
Weapons!!!...Batman (p53) Can Be
ACTIVATED!
Once p53 is phosphorylated
(Lucius Fox = p53 kinase
called Chk2), Batman is
ready to GO
Batman (p53) Is Signaled!!
-Increase in p53 levels (Batman fighting),
triggers p21Cip1 protein, a CDK inhibitor to
halt cell cycle advance (allow time to fight
crime)
- The weapons Lucius Fox supplies Batman
halt crime from happening in Gotham City
Who Is Mdm2 in the Dark Knight?
• The Joker
• Why?
• Goal is to disrupt Gotham City by creating
chaos
• Occupies Batman so he can’t protect the city
• Mdm2 prevents p53 from functioning
Who Is ARF in the Dark Knight?
• Helps Batman
• Attempts to catch the Joker
• Brings the Joker to jail
Officer / Commissioner Gordon
Activity 2 (5-10 min)
Complete the handout given at the start of
class (you may discuss with a partner) to
match molecules/cellular activities with
characters in the Batman movie
Activity 2 – Results
• Two Face: Mdm2 Promoter – Over expression allows Joker
to create more chaos
• Batman’s weapons: p21cip1 (CDK inhibitor)— Batman’s
weapons needed to fight crime
• Gotham: Genome—Fighting over control between Batman
and the Joker
• Bat Signal: ATM/ATR kinases— Signals Batman when crime
is occurring
• Gotham Police Department: Proteins that cause apoptosis,
thereby stopping DNA damage.
• Lucius Fox: Chk2 kinase—gives Batman his suit to activate
his capabilities to stop crime
• Crime alert system: Cell cycle checkpoints—Crime taking
place in Gotham city initiates Batman to take action
Functional p53 Triggers Apoptosis
• Apoptosis is a more drastic response than halting
the cell cycle
–
–
–
–
Plasma membrane herniates, forming blebs
Nucleus collapses
Cell breaks up into fragments
Neighboring cells ingest the fragments to recycle chemical
matter
What Triggers Apoptosis?
• Hypoxia
• DNA damage (many causes)
• Oncogene signaling
Apoptosis is triggered by various stressors.
Since so many things could go wrong, virtually
all human cancer cells should have
anti-apoptotic strategies to survive
Cancer Cell Strategies for Avoiding
Apoptosis
• Inactivation of the p53 pathway
– Mutated gene
– Altered promoter
– Inhibition of protein
• Overexpression of Mdm2
– Altered promoter
– Interaction with p53
Mutation of p53
• Li-Fraumeni syndrome was discovered in the early
1980s
• Found a group of families that showed increased
susceptibility to various cancers
• In 1990 found that in many of these cases, there was a
mutation in chromosome 17, where the p53 gene is
located
• In 70% of multicancer families, mutations in the alleles
of p53 found to be genetically inherited
• Seemed reasonable that mutations in p53 gene should
predispose a person to development of various types
of tumors
Mutation of p53
• p53 gene is altered in almost half of all
human cancer cell genomes
• Have found a variety of point mutations
scattered across the p53 reading frame
The Dark Knight
• What happens to Batman in the middle of the
movie?
• How does this relate to loss of p53 function?
Overexpression of Mdm2
• A small percentage of human tumors will
overexpress Mdm2
• Due to a mutation in the promoter region
• Excess Mdm2 proteins bind to p53 proteins
and prevent functional activation of apoptosis
The Dark Knight
Describe Mdm2 overexpression in terms of
the Joker.
The Joker has a group of men working with
him to fight Batman (p53) and stop its
function of protecting Gotham (genome)
Hyperactivation of Akt/PKB Pathway
• Activated by tyrosine kinase receptors and Ras
• PI3K is activated, increasing PIP3 and activation of
Akt/PKB
• Akt/PKB can phosphorylate pro-apoptotic proteins,
inhibiting them
Hyperactivation of Akt/PKB Pathway
• Akt/PKB can also cause Mdm2 to inhibit p53
• Akt/PKB kinase can phosphorylate Mdm2 at a
site that sends it to the cytoplasm to target p53
for degradation
Advantages Gained by Tumor Cells
• When p53 is mutated, there is limited apoptosis and
tumor cells can survive during hypoxia while the
blood supply is made
• p53 triggers apoptosis in response to DNA damage –
cells with damaged DNA have better survival rate,
increased gene alterations and accelerated tumor
development
Activity 3 – Role Play! (30 min)
Time to put together everything we have
learned today and characterize the Dark
Knight in terms of proteins involved in cancer
pathways.
Here are your movie characters:
Batman
Lucius Fox
Gordon
Joker
Harvey Dent/
Two Face
Activity 3
Scenario A:
Commissioner Gordon arrests the Joker and
brings him to the jail. How does this affect
Batman’s next action and significance to Gotham
City?
Scenario B:
Detective Gordon is thought to be dead. How
does his disappearance impact the conflict
between the Joker and Batman in Gotham City?
What would change if Two-Face joins the Joker?
Learning Objectives
1. Conclude the role of p53 in preserving genome
stability (Bloom’s Cognition Level 2, Understand)
2. Relate the functions of p53, MDM2, and Arf to
triggering apoptosis and cell cycle arrest (Bloom’s
Cognition Level 3, Apply)
3. Propose a mechanism for a cell’s response to a
situation in terms of a familiar story (Bloom’s
Cognition Level 6, Create)
Questions??
For next time
• Read research article:
Post SM, Quintás-Cardama A, Pant V, Iwakuma T,
Hamir A, Jackson JG, Maccio DR, Bond GL, Johnson
DG, Levine AJ, Lozano G. A high-frequency regulatory
polymorphism in the p53 pathway accelerates tumor
development. Cancer Cell. 2010 Sep 14;18(3):22030.
•
Pay close attention to the six figures in the article, and
be prepared to explain them or bring specific
questions about them, for discussion.
A High-Frequency Regulatory
Polymorphism in the p53 Pathway
Accelerates Tumor Development
Learning Objectives
1. Recall the functions of Mdm2 and p53 and how the
regulation of each affects apoptosis and cell cycle
arrest(Bloom’s Cognition Level 1, Remember)
2. Graph the expected results when the conditions of a
given experiment are altered (Bloom’s Cognition Level
3, Apply)
3. Invent an analogy to present biological data in a more
familiar context (Bloom’s Cognition Level 6, Create)
Review: Why Is Batman p53?
• Guardian of Gotham city (genome)
• Acts to prevent crime (cell cycle arrest), and
when necessary destroy evil (apoptosis)
• Unstable depiction of heroism
Activity 1 (10 minutes)
What are some other functions of p53? What
is its relationship with Mdm2? What can
trigger p53 inactivation in the p53/Mdm2
pathway?
New Material! Introduction
- Mdm2 amplified in >30% of sarcomas, over-expressed in human cancers with
wild type p53
- Mdm2 is a proto-oncogene, encodes E3 ubiquitin ligase that negatively
regulates p53 protein stability and transcriptional activity
- Mdm2 gene has two promoters. P1 controls “basal” expression and P2 is
regulated by many TFs including p53.
- Single Nucleotide Polymorphism (T-to-G) in P2 (SNP309G) yields homozygotes
(G/G) or heterozygotes (T/G)
- G/G mutation enhances binding of transcriptional activator Sp1 to P2
resulting in increase in Mdm2 transcription
Single Nucleotide Polymorphism
• A single nucleotide variation in a genetic sequence
that occurs at appreciable frequency in the
population
• 2 alleles differ in one location
• Common: 1 of every 300 nucleotides, on average
• Most often occur between genes, have no effect
-Increased levels of Mdm2, decreased levels of p53 ->
increased cancer risk!
-Mdm2-SNP309G associated with an increased cancer risk
in human tumors that express wild type p53, but not in cells
with mutant p53
- To understand mechanisms regulating this pathway during
tumorigenesis, most research focuses on mouse models
that genetically delete p53, over express genes that regulate
p53, or produce mutant proteins that mimic human
mutations
-This study used naturally occurring polymorphism in Mdm2
promoter to generate two humanized Mdm2 (SNP309G and
SNP309T) alleles to examine polymorphoism on tumor
development
Figure 1A: Generation of Mice Containing Either the Humanized
Mdm2-SNP309G (mutant) or Mdm2-SNP309T (control) Allele
- Generated the humanized allele constructs by replacing mouse
intron 1 containing P2 promoter with human intron 1 with G or T
polymorphism
- Black boxes are Mdm2 exons
- Mdm2 translation begins at exon 3, downstream from
construct
Figure 1B
- Southern Blot Analysis
revealed correct
targeting of both
constructs at Mdm2
locus
- Southern Blot Analysis
using probe verified
single copy integration
and indicated absence of
other insertions
Figure 1 Take Away
What was the take home message of
Figure 1?
- Wanted to see if they can replace Mdm2
alleles in mice with human alleles
(Mdm2-SNP309T and SNP309G)
Figure 2A: Mdm2-SNP309G/G Mice Have Higher
Levels of Mdm2 mRNA as Compared to
Mdm2SNP309T/T and C57Bl/6 Mice
- Real-time RT-PCR analysis
for Mdm2 mRNA levels in
spleen, brain, thymus, and
uterus of C57Bl/6 (C),
Mdm2SNP309G/G (G/G),
and Mdm2SNP309T/T (T/T)
mice
RNase protection assay
•
•
•
•
•
•
•
Purpose: to quantify amounts of specific RNA sequences
Lyse cell and obtain total RNA
Design antisense probe
Probe binds to mRNA of interest
Protects mRNA from degradation
Precipitate probe/mRNA complex
Use formaldehyde agarose gel to separate mRNA and quantify
Figure 2B
• RNase protection
analysis for Mdm2
mRNA levels in spleen
of Mdm2SNP309G/G
and Mdm2SNP309T/T
mice.
• Fold change
determined P2/P1
Conclusion: Increase in Mdm2SNP309G mRNA in mice arises primarily
from expression at the P2 promoter; this confirms the
overexpression effect of the mutation on the P2 promoter
Take Away Message From Figure 2
The data indicates that the increased levels
of Mdm2 in vivo are specifically due to a
mutation (SNP309G) of the Mdm2 gene in
the P2 promoter
Figure 3a: Mdm2 mRNA levels after
treatment with mithramycin A
• Mithramycin A disrupts
activity of Sp1, Tx = 500nM
18hr
– What is Sp1?
– Why would this affect mRNA
levels?
• RT-PCR analysis for Mdm2
RNA
• Compare the mRNA levels of
the Mdm2SNP309G/G and
Mdm2SNP309T/T mouse embryo
fibroblasts with no treatment
Figure 3b: Mdm2 protein levels after
treatment with mithramycin A
• Western blot analysis
– Used imaging software to
determine intensity of
bands
– Mdm2 and β-actin
antibodies
– What were the positive
and negative controls
used?
• What can you conclude
from this data?
Figure 3c: Different proliferation rates
• 3 cell lines per genotype
• Assayed for cell number
using absorbance values
at 1, 3, and 5 days
• What does the slope of
each line represent?
Figure 3 Take-Away
• Mdm2 RNA and protein levels were
significantly reduced in Mdm2SNP309G/G MEFs
with mithramycin A treatment
• Mithramycin A decreased expression of
Mdm2 only in Mdm2SNP309G/G cells
• Mdm2SNP309G/G cells had an increased rate of
proliferation compared to Mdm2SNP309T/T
Figures 4a and 4b: Mdm2 and p53
protein levels in 3 genotypes
Western blots
– Analyzed lysates from
spleens
– Mdm2, p53, and βactin antibodies
– Means of Mdm2 and
p53 were determined
by comparison to
normalized control
ratios
Ionizing Radiation
• Radiation when interacting with an atom, removes
tightly bound electrons from its orbit, causing the atom
to become charged or ionized
-Ex: alpha particles, beta particles, neutrons
• Exposure to IR leads to DNA damage
• Clustered DNA damage
- multiple DNA lesions induced
Figure 4c: Measurement of apoptosis
• Mice were exposed to
low dose IR
• Hematoxylin and
eosin stain – nucleus
is blue
• Added antibodies for
cleaved caspase 3
• Calculated the mean
of cells with cleaved
caspase 3
 What is the significance of this data?
Figure 4d: Quantification of
downstream p53 targets
• RT-PCR analysis for RNA levels of targets
• Puma = pro-apoptotic factor
• Ccng1 = cell cycle inhibitor
 Little change in cells without IR
 mRNA levels were lower in G/G with IR treatment
Figure 4e: Measurement of apoptosis
with mutant p53
• Compared Mdm2SNP309G/G and Mdm2SNP309T/T alleles with
heterozygous mutant p53 after low dose IR
• Significant reduction in G/G compared to T/T
 Diminished p53 response after DNA damage with both
wild-type and heterozygous mutant p53
Figure 4 Take-Away
• Spleens from Mdm2SNP309G/G mice had elevated levels of
Mdm2 and lower levels of p53
• Presence of 2 Mdm2SNP309G alleles significantly inhibited
p53-dependent apoptosis in response to DNA damage
in wild-type and heterozygous mutant p53 backgrounds
• Presence of Mdm2SNP309G allele attenuates activation of
the p53 pathway in the thymus after low dose IR
Learning Objectives
1. Recall the functions of Mdm2 and p53 and how the
regulation of each affects apoptosis and cell cycle
arrest(Bloom’s Cognition Level 1, Remember)
2. Graph the expected results when the conditions of a
given experiment are altered (Bloom’s Cognition Level
3, Apply)
3. Invent an analogy to present biological data in a more
familiar context (Bloom’s Cognition Level 6, Create)
Activity 2 (15 min)
Assume that the transcription of Mdm2 in
the G/G and T/T mice is not affected, but
we introduce an inhibitor of the
translation of Mdm2 protein.
How will the graphs in Figure 4 change?
Figure 4 graphs
a.
Protein levels of Mdm2 and p53
e.
c.
Apoptosis levels, wt p53
Apoptosis levels, mutant p53
Figure 5 A & B
• How do the mutations
affect survival rates?
– SNP309 G/G showed a lower
percent survival than T/T
mutation
– When coupled with p53
mutations, drastically
reduced survival
Figure 5 C &D
• Tumors expressing Mdm2 levels
Mdm2
Caspase
– Mutant promoter expressed
more Mdm2 than normal
– >10% of cells had to stain
positive for Mdm2 to be
significant
– Is 48% good enough?
• No significant
difference in
p53 LOH in
G/G vs. T/T
mice
Figure 6 A-K
• Different types of cancers apparent from p53
mice and Mdm2 mice
• Mammary adenocarcinomas not shown in
p53 cohorts
• D and I label estrogen receptors, placing the
tumor in breast
Conclusions
• What can we take away from this paper?
– Increase in Mdm2SNP309 G levels leads to
decreased p53 levels and decreased apoptosis
– Mdm2SNP309 G significantly increases cancer risk
and shortens survival time
– Cancers are observed in various tissues,
particularly breast cancer
– Even slight changes to Mdm2 levels can upset the
stability and activity of p53
Questions?
Learning Objectives
1. Recall the functions of Mdm2 and p53 and how the
regulation of each affects apoptosis and cell cycle
arrest(Bloom’s Cognition Level 1, Remember)
2. Graph the expected results when the conditions of a
given experiment are altered (Bloom’s Cognition Level
3, Apply)
3. Invent an analogy to present biological data in a more
familiar context (Bloom’s Cognition Level 6, Create)
Activity 3 (30 minutes)
• Now it’s time to put your understanding of
the paper to the test.
• Divide up into groups of no more than 4
• Construct the plot of a new Dark Knight
movie using the paper as the script.
– Discuss each figure as a scene in the movie.
– What should the audience take away from the
movie?
Answer to Question 3
• Figure 1: Two Face mutates and becomes evil, joins the Joker
• Figure 2: The presence of mutant Two Face increases the
ability of the Joker to suppress the activity of Batman
• Figure 3: When mutant Two Face is removed from the
equation, the Joker can’t create as much chaos. With mutant
Two Face active, chaos can accumulate much faster
• Figure 4: When the Joker and mutant Two Face are working
together Batman is overwhelmed and has a diminished effect.
Also Batman is not as good at protecting the city.
• Figure 5: With mutant Two Face, the city is more likely to
succumb to the chaos and be destroyed. When Batman is
injured the effects are worsened. Also, the Joker doesn’t have
to be everywhere for chaos to occur.
• Figure 6: There are different types of destruction in Gotham
when Two Face is present. Also more likely to have multiple
serious crime scenes when mutant Two Face is helping.
Answer to Question 3
• The Joker getting help from Two Face
overwhelms Batman, plunging the city into
chaos faster, especially if Batman is injured.