p53 and Control of the Cell Cycle

1
p53 and Control of the Cell Cycle
2
Learning Objectives

• Conclude the role of p53 in preserving genome
  stability (Blooms Cognition Level 2, Understand)
• 2. Relate the functions of p53, MDM2, and Arf to
  triggering apoptosis and cell cycle arrest
  (Blooms Cognition Level 3, Apply)
• 3. Propose a mechanism for a cells response to a
  situation in terms of a familiar story (Blooms
  Cognition Level 6, Create)

3
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

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Mutant Version of p53

• Knudsons 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?
5
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

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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.
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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

8
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)

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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 p53s function
• p53 acts as the guardian of the genome

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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

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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

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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

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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

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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

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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.

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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?vFXMB5KNYgMwfeature
related
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Activity 1 Results

• p53 Batman Guards the genome, protects Gotham
• Mdm2 Joker Inhibits p53, creates chaos by
  inactivating Batman
• ARF GordonBinds mdm2, prevents it from binding
  p53 helps Batman by jailing the Joker

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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

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Weapons!!!...Batman (p53) Can Be ACTIVATED!
Once p53 is phosphorylated (Lucius Fox p53
kinase called Chk2), Batman is ready to GO
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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

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Who Is Mdm2 in the Dark Knight?

• The Joker
• Why?

• Goal is to disrupt Gotham City by creating chaos
• Occupies Batman so he cant protect the city
• Mdm2 prevents p53 from functioning

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Who Is ARF in the Dark Knight?

• Helps Batman
• Attempts to catch the Joker
• Brings the Joker to jail

Officer / Commissioner Gordon
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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

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Activity 2 Results

• Two Face Mdm2 Promoter Over expression allows
  Joker to create more chaos
• Batmans weapons p21cip1 (CDK inhibitor)
  Batmans weapons needed to fight crime
• Gotham GenomeFighting 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 kinasegives Batman his suit to
  activate his capabilities to stop crime
• Crime alert system Cell cycle checkpointsCrime
  taking place in Gotham city initiates Batman to
  take action

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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

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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
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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

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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

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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

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The Dark Knight

• What happens to Batman in the middle of the
  movie?
• How does this relate to loss of p53 function?

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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

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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)
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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

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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

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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

Chaos!
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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.

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Here are your movie characters

• Batman Lucius Fox
• Gordon
• Harvey Dent/
• Joker Two Face

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Activity 3

• Scenario A
• Commissioner Gordon arrests the Joker and brings
  him to the jail. How does this affect Batmans
  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?

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Learning Objectives

• Conclude the role of p53 in preserving genome
  stability (Blooms Cognition Level 2, Understand)
• 2. Relate the functions of p53, MDM2, and Arf to
  triggering apoptosis and cell cycle arrest
  (Blooms Cognition Level 3, Apply)
• 3. Propose a mechanism for a cells response to a
  situation in terms of a familiar story (Blooms
  Cognition Level 6, Create)

40
Questions??
41
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 1418(3)220-30.
• Pay close attention to the six figures in the
  article, and be prepared to explain them or bring
  specific questions about them, for discussion.

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A High-Frequency Regulatory Polymorphism in the
p53 Pathway Accelerates Tumor Development
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Learning Objectives

• Recall the functions of Mdm2 and p53 and how the
  regulation of each affects apoptosis and cell
  cycle arrest(Blooms Cognition Level 1, Remember)
• 2. Graph the expected results when the conditions
  of a given experiment are altered (Blooms
  Cognition Level 3, Apply)
• 3. Invent an analogy to present biological data
  in a more familiar context (Blooms Cognition
  Level 6, Create)

44
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

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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?
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New Material! Introduction

• Mdm2 amplified in gt30 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

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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

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• Increased levels of Mdm2, decreased levels of p53
  -gt 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

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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

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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
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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)

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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
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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

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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
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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

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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

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Figure 3b Mdm2 protein levels after treatment
with mithramycin A

• Western blot analysis
• Used imaging software to determine intensity of
  bands
• Mdm2 and ß-actin antibodies
• What were the positive and negative controls
  used?
• What can you conclude from this data?

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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?

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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

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Figures 4a and 4b Mdm2 and p53 protein levels in
3 genotypes

• Western blots
• Analyzed lysates from spleens
• Mdm2, p53, and ß-actin antibodies
• Means of Mdm2 and p53 were determined by
  comparison to normalized control ratios

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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

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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?
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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
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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

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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

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Learning Objectives

• Recall the functions of Mdm2 and p53 and how the
  regulation of each affects apoptosis and cell
  cycle arrest(Blooms Cognition Level 1, Remember)
• 2. Graph the expected results when the conditions
  of a given experiment are altered (Blooms
  Cognition Level 3, Apply)
• 3. Invent an analogy to present biological data
  in a more familiar context (Blooms Cognition
  Level 6, Create)

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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?

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Figure 4 graphs
a.
Protein levels of Mdm2 and p53
e.
c.
Apoptosis levels, wt p53
Apoptosis levels, mutant p53
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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

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Figure 5 C D

• Tumors expressing Mdm2 levels
• Mutant promoter expressed more Mdm2 than normal
• gt10 of cells had to stain positive for Mdm2 to
  be significant
• Is 48 good enough?

Mdm2 Caspase

• No significant difference in p53 LOH in G/G vs.
  T/T mice

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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

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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

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Questions?
74
Learning Objectives

• Recall the functions of Mdm2 and p53 and how the
  regulation of each affects apoptosis and cell
  cycle arrest(Blooms Cognition Level 1, Remember)
• 2. Graph the expected results when the conditions
  of a given experiment are altered (Blooms
  Cognition Level 3, Apply)
• 3. Invent an analogy to present biological data
  in a more familiar context (Blooms Cognition
  Level 6, Create)

75
Activity 3 (30 minutes)

• Now its 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?

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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 cant 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 doesnt 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.

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Answer to Question 3

• The Joker getting help from Two Face overwhelms
  Batman, plunging the city into chaos faster,
  especially if Batman is injured.