PhIP-induced Mutation in
Mlh1-deficient Mice
Stacy Hill
6/28/2016
Stacy Hill Buermeyer Lab Group
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Significance
2-5% of all colorectal cancer cases in humans
result from a genetic predisposition called
hereditary nonpolyposis colorectal cancer
(HNPCC).
HNPCC is the most commonly inherited form
of colorectal cancer.
Those who have inherited HNPCC have an
80% risk of developing an early onset internal
cancer, such as colorectal cancer.
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HNPCC Syndrome & Mlh1
HNPCC is a syndrome caused by inherited defects in
DNA Mismatch Repair (MMR).
There are 4 different MMR genes linked to HNPCC,
Mlh1 and Msh2 being the most common.
Most HNPCC individuals have normal MMR activity in
their cells, due to the fact that they are
heterozygous for their MMR mutation.
In the rare event that an individual is born with no
proficient MMR activity, the individual usually dies
by the age of 5 years.
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HNPCC Syndrome
Individual cells that
become homozygous
deficient for Mlh1 are
at high risk for
becoming cancers.
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Risk Factors Of HNPCC
Little is known about the modulation of
cancer risk by environmental factors, such as
heterocyclic amines (HCA).
Mlh1-/- mice can be used to identify
modulators of cancer risk associated with
MMR deficiency.
Research Question: Does HCA exposure
actually increase the risk of cancer
development in an individual with MMR
deficiency?
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What Are Heterocyclic Amines?
Heterocyclic amines (HCA) are the
carcinogenic chemicals formed from the
cooking of muscle meats such as beef, pork,
fowl, and fish.
HCA form when amino acids and creatine (a
chemical found in muscles) react at high
cooking temperatures.
Frying, broiling, and barbecuing produce the
largest amounts of HCA because the meats
are cooked at very high temperatures.
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What Are We Afraid Of?
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)
PhIP is the most abundant
HCA, which we are currently
using for analysis.
MMR deficient cells have
been shown to be
hypermutable by exposure to
PhIP in cell culture.
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Experimental Model
We use genetically engineered mice to model cancer
predisposition in individuals with MMR deficiency.
Wildtype Mice


PhIP
Vehicle
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Mlh1-/- Mice


Stacy Hill Buermeyer Lab Group
PhIP
Vehicle
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Study Design
PhIP vs. Vehicle
Mutagenesis
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Tumorigenesis
Stacy Hill Buermeyer Lab Group
Cell Turnover
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Research Focus
Mutagenesis



The level of mutations, induced by PhIP, in both
wildtype (normal) mice and in Mlh1-/- mice.
Mlh1-/- or “knockout” mice are Mlh1-deficient.
Mutations resulting from PhIP exposure are
believed to be a result of the formation of PhIPDNA adducts, primarily to guanine residues.
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Characterizing PhIP-induced
Mutations
A recoverable lambda shuttle vector, previously
integrated into the mouse genome, will be used to
characterize mutations.
Illustration of the mouse chromosome.
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Recovering Lambda Phage
Isolating Genomic
DNA
Mutation
Selection
Phage
Packaging
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G1250 E.coli
infection
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The Selection Process
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Data Analysis
All of the plates are then collected and the
number of visible plaques, or plaque forming
units (pfu) are counted, to determine
mutation frequency (MF).
MF =
# of pfu 24ºC
‘
# of pfu 37ºC x Dilution Factor
Plaques are then purified and the cII gene is
sequenced. Then mutations are determined.
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Mutation Frequency
Results (Mutation Frequency)
1.20E-03
1.00E-03
8.00E-04
6.00E-04
Vehicle
PhIP
4.00E-04
2.00E-04
0.00E+00
Wildtype
Knockout
Treatment Group
PhIP-induced mutations:


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Wildtype
Knockout
6 x 10-5
47 x 10-5
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Results (cII Gene Map)
PhIP n=35 C
T
A T
T A
KO 5Х-ATGGTTCGTGCAAACAAACGCAACGAGGCTCTACGAATCGAGAGTGCGTTGCTTAACAAAATCGCAATGCTTGGAACTGAGAAGACAGCGGAAGCTGTGGGCGTTGATAAGTCGCAGATCA
Vehicle
A
AG
A
A
C
A
T
A
G
T
n=50
G
A
A
T
T
G
s t
T
s  tt
C
G
s 
PhIP n=35
T
T
t s  
A
C T
C
T
ss s
GCAGGTGGAAGAGGGACTGGATTCCAAAGTTCTCAATGCTGCTTGCTGTTCTTGAATGGGGGGTCGTTGACGACGACATGGCTCGATTGGCGCGACAAGTTGCTGCGATTCTCACCAATAAAAAACGC
Vehicle n=50
G
G
ss  
s G
T
T
s  
s  
s  
t 
s  
A
PhIP n=35
CCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGA -3Х
Vehicle n=50
PhIP n=12
A
T
T
A
A
T A
WT 5Х-ATGGTTCGTGCAAACAAACGCAACGAGGCTCTACGAATCGAGAGTGCGTTGCTTAACAAAATCGCAATGCTTGGAACTGAGAAGACAGCGGAAGCTGTGGGCGTTGATAAGTCGCAGATCA
Vehicle n=2
A
A
PhIP n=12
T
T
t
T
A
GCAGGTGGAAGAGGGACTGGATTCCAAAGTTCTCAATGCTGCTTGCTGTTCTTGAATGGGGGGTCGTTGACGACGACATGGCTCGATTGGCGCGACAAGTTGCTGCGATTCTCACCAATAAAAAACGC
Vehicle n=2
PhIP n=12
CCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGA -3Х
Vehicle n=2
t =independent insertion =non-independent insertion s =independent deletion =non-independent deletion
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Results (Mutation Spectrum)
Colon
Total Mutants
WT Vehicle
2
0 (0%)
Frameshifts
WT PhIP
12
KO Vehicle
50
1 (8%)
1
+G
-G
+A
-A
Other
KO PhIP
37
28 (56%)
3
11
19 (54%)
4
9
13
1?
6
2 (100%)
11 (92%)
22 (44%)
18 (46%)
Transitions
G:C  A:T
A:T  G:C
2
2
3
2
1
19
12
7
8
8
Transversions
G:C  C:G
G:C  T:A
A:T  C:G
A:T  T:A
0
8
3
2
1
10
5
5
Base Substitutions
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1
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Summary
Mlh1-/- mice were hypersensitive to PhIPinduced mutations in the colon.
Most PhIP-induced mutations in both wildtype
and knockout mice involved G/C base pairs.
Preliminary data suggest Mlh1-/- mice are
hypersensitive to PhIP-induced carcinogenesis.
PhIP, and perhaps other HCA, exposure
may increase cancer risk in people with
MMR deficiencies.
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Acknowledgements
Howard Huges Medical Institute (HHMI)
Undergraduate Research Innovation
Scholarship Creativity (URISC)
Stephanie Smith-Roe
Andrew Buermeyer
Kevin Ahern
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