DNA Mismatch Repair-Dependent
Suppression in Genotoxicity of
Complex Environmental
Carcinogenic Mixtures
Casey Kernan
Mentor: Dr. Andrew Buermeyer
Department: Environmental & Molecular Toxicology
Oregon State University
Colorectal Cancer (CRC)
• 2nd leading cause of cancer deaths worldwide
• CRC claims nearly 50,000 lives/year in U.S.
• American Cancer Society estimates 147,000
new cases by 2011
Hereditary
Sporadic
20%
80%
Lynch Syndrome (HNPCC)
• Autosomal dominant genetic condition
• Mutation in one or more of the 4 MMR genes:
MLH1 & PMS2 (MutLα) and MSH2 & MSH6 (MutSα)
MSH6/
3
MSH2
PMS2
MLH1
Mismatch Repair
A number of cellular pathways, processes and environmental
genotoxins interact to influence an individual’s susceptibility
and risk for developing cancer.
Apoptosis
Recognition of Mismatch
ATP
MSH2
MSH2
ADP
MSH6/
3
MSH6/
3
MLH1
PMS2
•DNA replication error rate = 1 mispairing / 104-105 basepairs
•MutS heterodimer locates and binds mismatch
•MutL heterodimer recruited and endonuclease activated
Excision of Mismatch
MSH6/
3
MSH2
RPA
hEXO1
hEXO1
PMS2
MLH1
•Exonuclease1 Activity: 5’  3’ directed
•RPA (Replication Protein A)
•Binds ssDNA, prevents degradation, promotes Polymerase δ,
Correction of Mismatch
Pol δ/ε
RPA
c
RFC
DNA
Ligase
c
Nick
PCNA = Proliferating Cell Nuclear Antigen
RFC = Replication Factor C
Pol δ/ε = DNA Polymerase Delta/Epsilon
PCNA
Mutator Phenotype
Mutations are a driving force
behind cancer development
Mutated
MMR genes
Aberrant
MMR proteins
MLH1
Enhanced
proliferation
Replication errors
bypass defective
MMR systems
Mutated cells divide
Mutations inactivate
tumor suppressor
genes and enable
onco-genes (APC gene)
•unchecked growth
•loss of apoptotic ability
•acquisition of metastic ability
•resistance to chemotherapeutic agents (6-TG, MNNG, 5-FU)
PAHs – The Environmental Influence
•
•
•
•
Mutagenic and carcinogenic - large nonpolar compounds
Exposure: diet, smoking, grilling food, fossil fuel processing
Metabolized forming highly reactive diol epoxides (DE)
Benzo[a]pyrene is metabolically activated to benzo[a]pyrene diol
epoxide (BPDE) which binds to DNA forming bulky DNA adducts
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
BPDE-Induced 6-TGR Mutant Frequency
in MMR-Proficient and -Deficient Cell Lines
Mutant Frequency
(x 10-5)
50
HCT116+hch2 (MLH1-)
HCT116+hch3 (MLH1+)
40
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.) General phenomena of PAH’s, in complex mixtures?
Hypothesis
•We hypothesize that results seen with HCT 116
lines do reflect differences in MMR status rather
than other potential known or unknown differences
in the cell lines.
-Verify using DLD1 cell lines proficient and
deficient in MSH6
•We hypothesize that MMR-dependent suppression
of BPDE-induced mutations represent a
phenomenon generalizable to other PAH’s, including
environmentally relevant complex mixtures.
BPDE-Induced Mutation
Forward mutations induced by exposure to PAH’s are measured
using the reporter gene hypoxanthine-guanine phosphoribosyl
transferase (HPRT) 657bp
HPRT+
HPRTHAT media –
5 passages
Clear pre-existing mutants
HPRT+
Grown 8 days to
insure no HPRT+
protein present
1 hour BPDE exposure
Doses: 0, 25, 50, 100 nM
Gene HPRTProtein HPRT+/-
HPRTHPRT- mutant cells survive in
6-Thioguanine selective media
Bulky PAH-DNA adducts
Cell Lines
MMR Proficient
MMR Deficient
HCT 116 + Ch3
HCT 116 + Ch2
WT
MLH1+
MLH1-
DLD1+Ch2
WT
MSH6+
DLD1
MSH6-
Mutant Frequency Calculation
MMR Proficient
MMR Deficient
MLH1
135,000 cells
6-TG selective media
few colonies
MLH1
300 cells
Non-selective media
~150 colonies
135,000 cells
6-TG selective media
more colonies
MF=6-TG resistant colonies formed/(PE x # of plates)
MF=mutant frequency
PE = plating efficiency
300 cells
Non-selective media
~150 colonies
Results: PAH-induced mutation in MSH6- deficient
cells, similar to previous MMR+ proficient cells
•Technical issue with low plating efficiency in MSH6+
Mutation Identification
1
1 2 v
3 4
5
9
6
7 8
10 11 12
4
trypsinized cloning disc
Centrifuge
Total RNA Purification
2
3
5
6
RNA  cDNA PCR sequence
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
Batch 3 PCR Products – HCT 116+2 HPRT Mutants
CONTROLS
Mutant:
45 18 44 6
13 22 24 26 27 49 50 80 81
HPRT
product
657bp
Spectrum of HPRT Mutations
Spectrum Key
Insertion of one nucleotide base
Deletion of one nucleotide base
25.0%
GC → TA transversion 58.3%
GC → CG transversion 4.2%
GC → AT transition
AT → GC transition
12.5%
Conclusion
Preliminary data suggest:
•BPDE-induced spectra in MLH1 deficient cells different
from spontaneous mutations
•Too soon to tell if induced spectra differs in MMR
proficient vs. deficient cell lines
Future Investigations
Continue mutant analysis on remaining clones:
•HCT 116+Ch2
•HCT 116+Ch3
•DLD1
•DLD1+Ch2
Complex environmental mixtures
•Mutant frequency
•Individual mutation analysis
Research Goals & Significance
Goals
•Understand MMR functions as well as genetic influences and
their combined role in normal responses to carcinogens
•Accurate evaluation of an individuals susceptibility and risk to
developing CRC
•Provide insight for more effective and practical CRC screening
methods
•Develop novel models for studying other genetic and
environmentally linked diseases
Acknowledgements
• Howard Hughes Medical Institute
• Environmental Health Sciences Center
• Dr. Andrew Buermeyer
– Jacki Coburn
– Fatimah Almousawi
– Kimberly Sarver
• Dr. Vidya Schalk
• Dr. Kevin Ahern, program coordinator