Presented by: Sarah Ferrer
Under the mentorship of Dr. Andrew Buermeyer of the OSU Environmental
and Molecular Toxicology Department


MMR deficiency linked to colorectal (and
other) cancer predisposition.
Lynch Syndrome causes premature cancer
occurrence and greater reoccurrence.

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
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.
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
Growth curves created over several dilutions and
compared to the characterized doubling times.
TK6
Growth
Curves
(8/3-8/7)
MT1
(8/7-8/11)
MT1Growth
GrowthCurves
Curves(8/7-8/11)
(8/3-8/7)
10
10
10
cells/ml)
(*10^6cells/ml)
Conc.
(*10^6
Conc.
cells/ml)
(*10^6
Conc.
cells/ml)
(*10^6
Conc.

1
11 0
00
0
20
20 20
20
40
40
40
40
60
60
80
80
60
60
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
0.001
Hours
from
beginning
Hours from
beginning
Hours
beginning
Hours from
from beginning
100
100
80
80
120
120
100
100
0.2*10^6
A=0.2*10^6
0.2*10^6
A=0.2*10^6
B=0.1*10^6
0.1*10^6
0.1*10^6
B=0.1*10^6
C=0.05*10^6
0.05*10^6
C=0.05*10^6
0.05*10^6
D=0.01*10^6
0.01*10^6
D=0.01*10^6
0.01*10^6

Percentage
Percentage
of Wells
of Wells
withwith
Visable
HPRT
Colonies
Mutants
(Plating
Hypoxanthine
guanine
phosphoribosyl
Percentage of Wells with HPRT
Mutants Present Before HAT
Efficiency)
transferase (HPRT) reporter gene.
100%
100%
100%
90%
90%
99%
80%
80%
Percent
Percent
60%
97%
60%
50%
50%
40%
40%
30%
30%
20%
Culture with 12 million cells
70%
RPMI w/ HAT media
96%
Percent
70%
98%
95%
not present
Cells Mutants
Mutants
not present
94%
Cells Mutants
present present
Mutants
93%
Normal RPMI media
92%
20%
6-TG
exposed
91% plate
10%
10%
90%
0%
0%
MT1
MT1 (no HAT)
MTI efficiency plate
Plating
Tk6
Cell Line MT1 (HAT)
TK6 (no HAT)
Cell Line
Cell Line and Media Type
TK6
TK6 (HAT)


Re-run previous experiment to determine MF.
Pick 6-TG resistant colonies and analyze for
types of mutations.
Normal HPRT+ cell
Cell with HPRT- DNA,
but HPRT+ proteins
Cell with HPRTDNA and proteins
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.




Dr. Andrew Buermeyer
Vidya Schalk
Kevin Ahern
Howard Hughes Medical Institute