DNA Mismatch Repair-Dependent
Suppression of Genotoxicity of
Benzo[a]pyrene Diol Epoxide
Casey Kernan1, Dr. Andrew Buermeyer2
Department of BioResource Research1
Department of Environmental and Molecular Toxicology2
Oregon State University, Corvallis, Oregon
Research Aims
Cancer Risk
Source: National Institute of Environmental Health Sciences
Cancer – Global Killer
Figure Source: Executive Healthcare Management
Colorectal Cancer (CRC)
• 2nd leading cause of cancer deaths worldwide
• CRC claims nearly 50,000 lives/year in U.S.
• American Cancer Society estimates 141,210 new cases in the year
2012
CRC Incidence
• Risk Factors:
• Diet
• Environmental
exposure
• Genetic deficiencies
• Tobacco/alcohol use
• Industrialization
• Family history
• Physical inactivity
• Diabetes
• Sexually transmitted
HPV
Figure Source: GLOBOCAN 2008.
CRC Incidence/Mortality Rates
Colorectal
Colorectal Cancer
Cancer
Incidence
Death Rates*
Rates*
byby
Race/Ethnicity
Race/Ethnicity
and
and
Sex,
Sex,
U.S.,
U.S.,
1999–2007
1999–2007
Figure Source: Center for Disease Control and Prevention
Mismatch Repair (MMR)
Provides several highly conserved genetic stabilization
functions
4 Phases
1. recognition of mismatch (MutSα: MSH2/MSH6)
Strand discrimination (MutLα: PMS2/MLH1)
2. recruitment of repair enzymes
3. excision of incorrect sequence or base (Exo)
4. resynthesis by DNA polymerase
Mutator Phenotype
Mutations are a driving force behind cancer development
Mutated
MMR genes
Aberrant
MMR
proteins
Replication errors
bypass defective
MMR systems
MLH1
Enhanced
proliferation
Mutated cells
divide
Mutations
inactivate tumor
suppressor genes
and enable oncogenes (APC gene)
•unchecked growth
•loss of apoptotic ability
•acquisition of metastic ability
•resistance to chemotherapeutic agents (6-TG, MNNG, 5-FU)
Mutator Phenotype in Mouse Model
Heterozygous and homozygous Mlh1 mutant mice had
reduced longevity compared to their wild-type littermates
Source: Edelmann, 1999
Lynch Syndrome (HNPCC)
 Autosomal dominant genetic condition
 Mutation in one or more of the 4 MMR genes: MLH1 & PMS2
(MutLα) and MSH2 & MSH6 (MutSα)
MutLα
MutSα
Lynch Syndrome (HNPCC)
 Microsatellite = di, tri and mononucleotide repeats, occurs tens
of thousands of times across the genome
 MSI identified in >90% of CRC cases that arise in individuals
with Lynch Syndrome
PAHs – The Environmental Influence
 Mutagenic and carcinogenic - large nonpolar compounds
 Exposure: diet, smoking, grilling food, fossil fuel processing
 Benzo[a]pyrene -> benzo[a]pyrene diol epoxide (BPDE)
Benzo[a]Pyrene Metabolism
CYP1A1
Epoxidation
Oxidation
Quinones,
Phenols
Big Question
Global Hypothesis
Specific combinations of environmental exposures and cellular deficiencies
interact to influence cancer risk in individuals
Specific Hypothesis
MMR is a key pathway for reducing deleterious consequences (mutations)
from PAH exposure
Prediction
Cells lacking MMR will show increased PAH-induced mutation
Research Questions
BPDE-Induced 6-TGR Mutant Frequency
in MMR-Proficient and -Deficient Cell Lines
Mutant Frequency
(x 10-5)
50
HCT116+hch2 (MLH1-)
29.8±7 mutants/100 nM BPDE
40
HCT116+hch3 (MLH1+)
9.6±4 mutants/100 nM BPDE
30
20
10
0
50
-10
100
150
200
250
BPDE (nM)
3 Questions
1.) General phenomena of MMR-deficiency?
2.) What are the extra mutations induced?
3.) How would cells respond exposed to the parent compound
B[a]P and an exogenous metabolizing source?
Hypothesis
We hypothesize that preliminary results
with the MLH1-proficient and –deficient
cell lines reflect differences in MMR
status rather than other potential known
or unknown differences in the cell lines.
To test this hypothesis, I measured
BPDE-induced mutation rates in the
parent cell line HCT 116, and in another
genetically matched MSH6 proficient and
deficient set.
Cell Lines
MMR Proficient
HCT 116 + Ch3
WT
MLH1+
DLD1+Ch2
WT
MSH6+
MMR Deficient
HCT 116
MLH1-
DLD1
MSH6-
Overview: BPDE-Induced Mutation
HPRT+
HPRTHAT media –
5 passages
Clear pre-existing
mutants
Cytotoxicity
Plating
HPRT+
Phenotypic
HPRTExpression (8
days)to insure
HPRT- mutant cells survive in no HPRT+ protein
6-Thioguanine selective media present
1 hour BPDE exposure
Doses: 0-100nM dissolved
in DMSO
Gene HPRTProtein HPRT+/-
Bulky PAH-DNA adducts
Mutant Frequency Calculation
MMR
Proficient
MMR Deficient
MLH1
135,000 cells
MLH1
300 cells
6-TG selective media
media
Non-selective
135,000 cells
6-TG selective media
media
P.E.
few colonies
~150 colonies
300 cells
Non-selective
P.E.
more colonies
~150 colonies
MF=6-TG resistant colonies formed/(PE x # of plates x #cells plated)
MF= mutant frequency
PE = plating efficiency
DLD1 and DLD1+Ch2
BPDE-induced HPRT mutation in MSH6-proficient
and -deficient cells
Mutant Frequency
(x10-5)
60
DLD1 (MSH6-)
6.0±1 mutants/100 nM BPDE
DLD1+Ch2 (MSH6+)
34.9± mutants/100 nM BPDE
40
20
Conclusion: not clear
0
0
50
100
•Technical issue with low plating
efficiency
150
BPDE Dose (nM)
DLD1 Plating Efficiency
Plating Efficiency (%)
80
DLD1 (MSH6-)
DLD1+Ch2 (MSH6+)
60
•Possible mix-up with cell lines
•MSH3 possibly compensating
40
•No opportunity to follow-up
20
0
0
50
100
BPDE Dose (nM)
150
Initial Experiment – HCT 116
Mutant Frequency
(x 10-5)
150
BPDE-induced HPRT mutation in
HCT 116 Cells
100
50
0
0
50
100
150
BPDE Dose (nM)
• High level of spontaneous mutants rate, apparent in 0 dose
• Limits ability to detect and accurately measure BPDE-induced mutants
Problem:
High level of spontaneous mutants
• Insufficient HAT
• Excessive phenotypic expression
• Difference between HCT 116 and HCT 116+Ch2
HAT Treatment
Mutant Frequency in
HAT Passaged HCT 116 Lines
Mutant Frequency
(x10-5)
80
HCT 116+Ch2
HCT 116-B
HCT 116-K
60
40
20
0
0
1
2
3
4
5
6
7
8
HAT Passage #
HAT Passaged HCT 116 Lines
Plating Efficiency (%)
200
HCT 116+Ch2
HCT 116-B
HCT 116-K
150
100
50
0
0
1
2
3
4
5
HAT Passage #
6
7
8
Conclusion:
Five 1:5 HAT passages
sufficient to reduce
spontaneous, preexisting
mutants to frequency below
approximately
≤0.37x10-5 mutants
Phenotypic Expression
Spontaneous Mutant Induction in
Mutator Cell Line
Mutant Frequency
A
Limit of Detection based on plating conditions
T0
TA
B
Population Doublings
HAT
BPDE Exposure
Phenotypic expression - Time or number of population doublings following
BPDE exposure necessary to reduce level of preexisting HPRT protein in a
newly genetically mutant cell to levels that will not interfere with 6-TG
selection.
Phenotypic Expression
7 vs. 10 Days
BPDE-Induced Mutant Frequency
in HCT 116+Ch2 Cell lines
Mutant Frequency
(x 10-5)
60
7 day phenotypic
expression
10 day phenotypic
expression
40
20
0
0
50
100
150
200
250
BPDE Dose (nM)
HCT 116+Ch2
Plating Efficiencies
Plating Efficiency (%)
Results: no dramatically
different level of spontaneous
mutation with 7 vs. 10 days
phenotypic expression
50
7 day phenotypic
expression
40
10 day phenotypic
expression
30
20
10
0
0
50
100
150
BPDE Dose (nM)
200
250
Low plating efficiency in 7
day data might be
overestimating mutant
frequencies
Conclusion: need additional
experiment to resolve
significance of length of
phenotypic expression period
HCT 116 B vs. HCT 116 K
Mutant Frequency
(x 10-5)
BPDE-Induced Mutant Frequency
in HCT 116 K, HCT 116 B and HCT 116+Ch3
Cell Lines
Mutation Rates (mutants/100 nM BPDE):
MMR-deficient HCT 116 B
MMR-deficient HCT 116 K
MMR-proficient HCT
116+Ch3
150
100
HCT 116-B
HCT 116-K
HCT 116+Ch3
58.1±7
30.2±5
9.6±4
50
0
0
50
100
150
200
250
BPDE Dose (nM)
HCT 116 B and HCT 116 K
Plating Efficiencies
Plating Efficiency (%)
50
HCT 116-B
HCT 116-K
40
30
20
10
0
0
50
100
150
BPDE Dose (nM)
200
250
Conclusion: Finding is consistent with
preliminary data from Buermeyer
laboratory, demonstrating increased
BPDE-induced mutation in MLH1deficient vs. proficient cell lines
Conclusions
•Five 1:5 passages in HAT sufficient to reduce spontaneous, preexisting
mutants to frequency below approximately ≤0.37x10-5 mutants
•No dramatic difference in level of spontaneous mutation with 7 vs. 10 days
phenotypic expression time
•HCT 116-K and HCT 116-B mutation rate findings consistent with
preliminary data, demonstrating increased BPDE-induced mutation in
MLH1-deficient vs. proficient cell lines
Next Question
BPDE-Induced 6-TGR Mutant Frequency
in MMR-Proficient and -Deficient Cell Lines
Mutant Frequency
(x 10-5)
50
HCT116+hch2 (MLH1-)
29.8±7 mutants/100 nM BPDE
40
HCT116+hch3 (MLH1+)
9.6±4 mutants/100 nM BPDE
30
20
10
0
50
-10
100
150
200
250
BPDE (nM)
3 Questions
1.) General phenomena of MMR-deficiency?
2.) What are the extra mutations induced?
3.) How would cells respond exposed to the parent compound
B[a]P and an exogenous metabolizing source?
Mutation Identification: Colony -> RNA
1
5
9
2
6
10
3
7
4
8
11 12
trypsinized cloning
disc
Total RNA Purification
Centrifuge
1
4
2
5
3
6
Mutation Identification: RNA -> cDNA
Reverse Transcriptase PCR
cDNA
PCR – amplify HPRT gene
Primers
P3: -36 to -17
P4: 701 to 682
5’ CCTGAGCAGTCAGCCCGCGC 3’
5’ CAATAGGACTCCAGATGTTT 3’
Sequencing
agarose gel electrophoresis
Agarose Gel Electrophoresis
Representative PCR Products – HCT 116+2 HPRT
Mutants
CONTROLS
Mutant:
45 18 44 6
13 22 24 26 27 49 50 80 81
HPRT
product
737bp
Spectrum of Induced HPRT Mutations
Spectrum Key
Insertion of one nucleotide base
Deletion of one nucleotide base
13.6%
GC → TA transversion 63.6%
GC → CG transversion 4.5%
GC → AT transition
AT → GC transition
18.3%
Spontaneous HPRT Mutations
•Frameshift mutations observed:
(2) 213G insertion
(1) A499 deletion
•Typically expect ~50% frameshift with
spontaneous HPRT mutation in MMR-deficient
cells
•Limited analysis of spontaneous spectrum
Spectrum of Mutation Conclusions
Preliminary data suggest:
•BPDE-induced spectrum in MLH1-deficient cells is
different from spontaneous mutations
•BPDE-induced spectrum consistent with
adduction to guanine
•Too soon to tell if induced spectra differs in MLH1proficient vs. MLH1-deficient cell lines
Experiment 3
3 Questions
1.) General phenomena of MMR-deficiency?
2.) What are the extra mutations induced?
3.) How would cells respond exposed to the parent compound
B[a]P and a metabolizing reagent?
S9 Metabolizing Fraction
Induced P450 Enzymes
Aroclor 1254
S9 Fraction
• Phase I and II
enzymes
• Needs co-factors
Cytotoxicity with S9
S9 Metabolizing System
Cytotoxicity
Cell Survival (%)
15
Deficient HCT 116+Ch2
Proficient HCT 116+Ch3
10
5
0
0.0
0.5
1.0
1.5
2.0
2.5
B[a]P Dose (ug)
• Very Low Cell Survival, even with no B[a]P (0 dose) vs. typical
plating efficiencies
Mutant Frequency with S9
Mutant Frequency (x105)
HCT 116+2/3 - B[a]P S9 Fraction
50
Proficient HCT 116+Ch3
Deficient HCT 116+Ch2
40
30
20
10
0
0.0
0.5
1.0
1.5
B[a]P Dose (ug)
2.0
2.5
Summary
• Apparent high cytotoxicity with S9 metabolizing extracts
alone
• No apparent induction of HPRT mutations
• Need to optimize metabolizing environment conditions before
proceeding with complex mixtures
Future Investigations
Continue mutant analysis on remaining clones:
•HCT 116+Ch2
•HCT 116+Ch3
•DLD1
•DLD1+Ch2
•HCT 116+Ch2(S9)
•HCT 116+Ch3(S9)
Complex environmental mixtures
B[a]P equivalents
•Phenotypic Expression
•Optimize S9 Conditions
•Spectrum of Mutation
Research Goals and Significance
• Understand MMR functions as well as genetic influences and
their combined role in normal responses to carcinogens
• Accurate evaluation of an individual’s susceptibility and risk to
developing CRC
• Provide insight for more effective CRC screening methods
• Develop novel models for studying other genetic and
environmentally linked diseases
Acknowledgements

Dr. Andrew Buermeyer

Jacki Coburn

Fatimah Almousawi

Kimberly Sarver

Dr. Vidya Schalk

Dr. John Hays

Bioresource Research Program

Wanda Crannell

Dr. Kate Field

Howard Hughes Medical Institute

National Institute of Environmental Health Sciences

Environmental Health Sciences Center