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

Relevance of Research

MMR deficiency linked to colorectal (and other) cancer predisposition.

Lynch Syndrome causes premature cancer occurrence and greater reoccurrence.

Background: Mismatch Repair

MMR protects against DNA mutations.

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

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.

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

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

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

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

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.

Acknowledgements

Dr. Andrew Buermeyer Vidya Schalk Kevin Ahern Howard Hughes Medical Institute