Walter Piper
Dr. Andrew Buermeyer
Department of Environmental and Molecular
Toxicology
Oregon State University
2009 HHMI Undergraduate Research Program
Blue DNA – by Christopher Escabarte
http://yethzart.deviantart.com/art/Blue-DNA-75781660
©2008-2009 ~yethzart
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Alcohol (+80%-250%)
Smoking (+18%)
BMI (Body Mass Index)
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Figure 1 – Colon Cancer and Polyp
http://images.medicinenet.com/images/ILLUSTRATIONS/colon_
cancer.jpg
MedicineNet.com
(+50% in men when
BMI>25. Less risk in
women)
Diet
Genetic influence (+200%)
A 1st degree relative with
the cancer is one of the
biggest risk factors.
 The exact causes of this
genetic risk are unknown.
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Hereditary
Sporadic
Cases of Colorectal Cancer by type
5%
Hereditary
Sporadic
95%
Figure 2 – Percentage of total cases of
CRC by type.
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Current CRC risk evaluation  family history
Cytotoxicity assay  physical basis to evaluate
risk
Uses:
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Large scale studies
Individual patient evaluation
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DNA mismatch repair (MMR) is
a complex pathway involved in
the maintenance of DNA
integrity.
In the event that this repair
process fails, a mutation will
occur, which may lead to cancer.
MMR deficiency is linked to
hereditary colorectal cancer.
Figure 3 – DNA Mismatch Repair
http://www.rndsystems.com/mini_review_detail_objectname_MR03_DNADamageResponse.a
spx
R&D Systems
Moderate deficiencies present in the
normal range of the population
contribute to the genetic risk for
sporadic CRC.
 How can we accurately assess MMR
proficiency?
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 Genetically
 Biochemically
 Cell-based cytotoxicity assay
Figure 4 MMR proteins in DNA-repair and DNA-damage-response pathways
Biochemical Society Transactions
www.biochemsoctrans.org
(2005) 33, 689-693
Biochem. Soc. Trans.
Figure 5 – MNNG induced methylation
and attempted mismatch repair leading
to apoptosis.
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Use MTS to measure the relative
surviving cell fraction following
exposure to MNNG.
Relative cell survival should correlate
with the level of MMR proficiency.
Does MMR proficiency correlate with the
genetic risk for sporadic CRC?
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Cell survival is then measured using an MTS
Assay.
Cells biochemically reduce the tetrazolium
compound, MTS, into a colored formazan product
that has an absorbance maximum at 490 nm.
The amount of formazan dye is determined by
absorption of 490 nm UV light in a plate reader.
Figure 6 – General reaction for
the reduction of a tetrazolium to
a formazan.
http://en.wikipedia.org/wiki/F
ile:Tetrazolium_reduction.png
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We are using immortalized lymphoblasts from
blood samples to measure varying degrees of
MMR proficiency in the general population.
Differing degrees of MMR proficiency can be
characterized by consistent results showing a
distinct level of cell survival.
Is the assay sensitive and precise enough to
measure the slight differences in MMR
proficiency?
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Culture human lymphoblastoid cells
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Growth concentration: 50,000 – 1,000,000 cells/ml
Doubling time: ~21 hours
Treat cells in 96-well plates with the harmful
mutagen MNNG
Cell cultures that are more MMR proficient
undergo higher rates of apoptosis because they
have a more effective DNA damage signaling
pathway.
MTS assay is used to measure relative cell
survival.
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Currently establishing conditions for cytotoxicity
measurement using MTS assay, so an accurate cell
count can be determined based on signal reading.
What kind of media is best? Standard RPMI media,
media without phenol red, or media buffered with 25
mM HEPES?
Signal vs. Cell number at 2.5 hours
2.5
2
N-media
1.5
Signal
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P-media
1
B-media
0.5
Figure 7 – Signal in plate
reader vs. number of cells
in a well.
0
0
50000
100000
150000
200000
Number of cells
250000
300000
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Validate assay with initial set of clinical
samples.
Study the normal range of MMR proficiency in
the general population.
Large-scale studies could also be done to
definitively link MMR to sporadic CRC risk.
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Howard Hughes Medical Institute
Dr. Andrew Buermeyer
Vidya Schalk
Sarah Ferrer
Anneka Wickramanayake
Dr. Kevin Ahern