![Page 1: Presented by: Sarah Ferrer Under the mentorship of Dr. Andrew Buermeyer of the OSU Environmental and Molecular Toxicology Department](https://reader035.vdocuments.net/reader035/viewer/2022072006/56649f455503460f94c66aec/html5/thumbnails/1.jpg)
Mismatch Repair Responses to Carcinogenic Polycyclic Aromatic Hydrocarbons
Presented by: Sarah FerrerUnder the mentorship of Dr. Andrew Buermeyer of the OSU Environmental and Molecular Toxicology Department
![Page 2: Presented by: Sarah Ferrer Under the mentorship of Dr. Andrew Buermeyer of the OSU Environmental and Molecular Toxicology Department](https://reader035.vdocuments.net/reader035/viewer/2022072006/56649f455503460f94c66aec/html5/thumbnails/2.jpg)
Relevance of Research
MMR deficiency linked to colorectal (and other) cancer predisposition.
Lynch Syndrome causes premature cancer occurrence and greater reoccurrence.
![Page 3: Presented by: Sarah Ferrer Under the mentorship of Dr. Andrew Buermeyer of the OSU Environmental and Molecular Toxicology Department](https://reader035.vdocuments.net/reader035/viewer/2022072006/56649f455503460f94c66aec/html5/thumbnails/3.jpg)
Background: Mismatch Repair
MMR protects against DNA mutations.
![Page 4: Presented by: Sarah Ferrer Under the mentorship of Dr. Andrew Buermeyer of the OSU Environmental and Molecular Toxicology Department](https://reader035.vdocuments.net/reader035/viewer/2022072006/56649f455503460f94c66aec/html5/thumbnails/4.jpg)
PAHs are carcinogens found in the environment.
Metabolized by the liver and colon into diol epoxides.
PAHs used in my research project: Benzo-[a]-pyrene Benzo-[c]-phenanthrene
MMR proteins can bind to and recognize PAH adducted DNA
Background: Carcinogenic Polycyclic Aromatic Hydrocarbons
![Page 5: Presented by: Sarah Ferrer Under the mentorship of Dr. Andrew Buermeyer of the OSU Environmental and Molecular Toxicology Department](https://reader035.vdocuments.net/reader035/viewer/2022072006/56649f455503460f94c66aec/html5/thumbnails/5.jpg)
Hypothesis
MMR proficient cells are more effective at maintaining DNA integrity in
human lymphoblastic cell lines than MMR deficient cells when exposed to PAHs benzo-[a]-pyrene and benzo-
[c]-phenanthrene.
![Page 6: Presented by: Sarah Ferrer Under the mentorship of Dr. Andrew Buermeyer of the OSU Environmental and Molecular Toxicology Department](https://reader035.vdocuments.net/reader035/viewer/2022072006/56649f455503460f94c66aec/html5/thumbnails/6.jpg)
Method: Cell Culture
Human lymphoblastoid cell lines TK6 and MT1
Maintained under the following conditions: 10% complete RPMI media Incubated with 5% carbon dioxide and at
38°C Cell density between 5 x 104 cells/mL
and 1 x 106 cells/mL
![Page 7: Presented by: Sarah Ferrer Under the mentorship of Dr. Andrew Buermeyer of the OSU Environmental and Molecular Toxicology Department](https://reader035.vdocuments.net/reader035/viewer/2022072006/56649f455503460f94c66aec/html5/thumbnails/7.jpg)
0 20 40 60 80 100 1200.001
0.01
0.1
1
10
MT1 Growth Curves (8/7-8/11)
A=0.2*10^6
B=0.1*10^6
C=0.05*10^6
D=0.01*10^6
Hours from beginning
Co
nc.
(*1
0^6
cells
/ml)
0 10 20 30 40 50 60 70 80 90 1000.01
0.1
1
10
TK6 Growth Curves (8/3-8/7)
0.2*10^6
0.1*10^6
0.05*10^6
0.01*10^6
Hours from beginning
Co
nc.
(*1
0^6
cell
s/m
l)
0 20 40 60 80 100 120
0.01
0.1
1
10
TK6 Growth Curves (8/7-8/11)
A=0.2*10^6
B=0.1*10^6
C=0.05*10^6
D=0.01*10^6
Hours from beginning
Co
nc.
(*1
0^6
cells
/ml)
Growth curves created over several dilutions and compared to the characterized doubling times.
0 10 20 30 40 50 60 70 80 90 1000.01
0.1
1
10
MT1 Growth Curves (8/3-8/7)
0.2*10^6
0.1*10^6
0.05*10^6
0.01*10^6
Hours from beginning
Co
nc.
(*1
0^6
cell
s/m
l)
Method: Growth Curve Characterization
![Page 8: Presented by: Sarah Ferrer Under the mentorship of Dr. Andrew Buermeyer of the OSU Environmental and Molecular Toxicology Department](https://reader035.vdocuments.net/reader035/viewer/2022072006/56649f455503460f94c66aec/html5/thumbnails/8.jpg)
MT1 (no HAT) TK6 (no HAT) MT1 (HAT) TK6 (HAT)0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Percentage of Wells with HPRT Mutants
Mutants not presentMutants present
Cell Line and Media Type
Perc
ent
MTI TK60%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Percentage of Wells with Visable Colonies (Plating Efficiency)
Cells not presentCells present
Cell Line
Perc
ent
Hypoxanthine guanine phosphoribosyl transferase (HPRT) reporter gene.
Method: HPRT Gene Spontaneous Mutant Frequency Characterization
Culture with 12 million cells
Plating efficiency plate
6-TG exposed plate
RPMI w/ HAT media
Normal RPMI media
MT1 Tk60%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Percentage of Wells with HPRT Mutants Present Before HAT
Mutants not presentMutants present
Cell Line
Perc
ent
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Re-run previous experiment to determine MF.
Pick 6-TG resistant colonies and analyze for types of mutations.
Method: PAH Exposed HPRT Mutant Frequency
Normal HPRT+
cell
Cell with HPRT- DNA, but HPRT+ proteins Cell with HPRT-
DNA and proteins
![Page 10: Presented by: Sarah Ferrer Under the mentorship of Dr. Andrew Buermeyer of the OSU Environmental and Molecular Toxicology Department](https://reader035.vdocuments.net/reader035/viewer/2022072006/56649f455503460f94c66aec/html5/thumbnails/10.jpg)
Prediction
When exposed to benzo-[a]-pyrene and benzo-[c]-phenanthrene, MMR deficient cells lines exhibit a higher mutant frequency in the HPRT gene than MMR proficient cells.
![Page 11: Presented by: Sarah Ferrer Under the mentorship of Dr. Andrew Buermeyer of the OSU Environmental and Molecular Toxicology Department](https://reader035.vdocuments.net/reader035/viewer/2022072006/56649f455503460f94c66aec/html5/thumbnails/11.jpg)
Acknowledgements
Dr. Andrew Buermeyer Vidya Schalk Kevin Ahern Howard Hughes Medical Institute