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DNA Damage Response between Pluripotent and Differentiated Cells from Ataxia Telangiectasia Subjects
Lucy LinRonald P. Hart, Ph.D.
Department of Cell Biology and NeuroscienceRutgers, The State University of New Jersey
Robert Wood Johnson Medical School Summer Undergraduate Research Program 2014
Ataxia TelangiectasiaNeurodegenerative, inherited
disease caused by defect in ATM gene◦Causes tumor formation◦Impairs cerebellum◦Prevents repair of broken DNA
Kane, K.S. et al. 2002
Tumors from A-T
Cells that can be reprogrammed from adult somatic cells through transcription factors to become pluripotent
Induced Pluripotent Stem Cells
Sigma-Aldrichhttp://www.sigmaaldrich.com/life-science/stem-cell-
biology/ipsc.html
G1-S G2
ATM Protein Activates Chk2
Becker, T. and Haferkamp, S., 2013 (Modified by Lucy Lin)
SC1 Cells (control)◦ Both alleles of ATM
gene are normalCAR3 Cells (carrier)
◦ Deletion causing frameshift on one allele and one normal allele
Q3 Cells (A-T)◦ Compound
heterozygote: deletion causing frameshift on one allele and point mutation on other
Lazaropoulos, M., unpublished
CAR3 and Q3 ATM Allele Mutations
Protein Genotype
Protein Activity
ATM Mutations are Expressed in CAR3 and Q3 iPS Cell Lines
0.00
0.10
0.20
0.30
0.40
0.50
SC1 (Control) CAR3 (Carrier)Q3 (A-T)
iPS Cell LineN
orm
ali
ze
d I
n-
teg
rate
d D
en
sit
y
(AT
M B
an
d:B
eta
-A
cti
n B
an
d)
0.00
0.10
0.20
0.30
0.40
0.50
SC1 (Control) CAR3 (Carrier) Q3 (A-T)
iPS Cell Line
No
rma
lize
d I
nte
-g
rate
d D
en
sit
y
(53
BP
1 B
an
d:B
eta
-A
cti
n B
an
d)
Initial Hypothesis DNA damage response is
mediated by ATM in SC1 cells, while there is a lack of ATM mediation in CAR3 and Q3 cells
◦ In SC1 control iPS cells (ATM present): most number of puncta after radiation treatment
◦ In Q3 A-T case iPS cells (both ATM alleles are mutated): least number of puncta after radiation treatment
Introduce DNA damage to cells through ionizing radiation to investigate the effects on DNA damage repair capability in the SC1, CAR3, and Q3 iPSCs
DNA Damage Assay of iPSCs
No Significant Difference in DNA Damage Response in iPSCs
SC1 (Control) CAR3 (Carrier)
Q3 (A-T)0
10
20
30
40
50
60
70
80
No IrradiationIrradiation (1 Gy)
Mean
gH
2A
.X A
rea (
mic
rom
-ete
rs^
2)
p = 0.155
p = 0.311 p = 0.254
ATR Protein Activates Chk1
G1-S G2
Becker, T. and Haferkamp, S., 2013 (Modified by Lucy Lin)
ATR Protein is Expressed in iPS Cell Lines
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
SC1 (Control) CAR3 (Carrier)Q3 (A-T)
iPS Cell Line
Norm
alized
In
teg
rate
d
Den
sit
y
(ATR
Ban
d/B
eta
-Acti
n B
an
d)
No Significant Change in gH2A.X Levels in Mouse Embryonic Stem Cells
Figure 3C. Serrano et al. , 2011
DNA damage response is mediated by ATR in pluripotent cells
Once differentiated, ATM mediates DNA damage response
New Hypothesis
SC1 (Con-trol)
CAR3 (Carrier)
Q3 (A-T)0
20000
40000
60000
80000
100000
120000
140000
No Irradiation Irradiation (1 Gy)
GABA iN Cell Line
Are
a (
pix
els
) X
Mean
Gra
y
Valu
e
Changes in gH2A.X Expression in Control GABA Neurons
SC1 NI
SC1 IR
CAR3 NI
CAR3 IR
Q3 NI
Q3 IR
gH2A.X
Hoechst
NI = No irradiationIR = Irradiation
p = 7.75X10-
6
****
p = 0.092 p =
0.115
No Irradiation
Irradiation (1 Gy)
Inhibition of ATM in SC1-Derived Embryoid Body Cells Results in Little Change in gH2A.X Levels
Vehicle Control (DMSO)
ATM Inhibition
(KU-55933)
ATR Inhibition (AZ-20)
ATM and ATR
Inhibition
gH2A.X gH2A.XHoechst HoechstNo Irradiation Irradiation
G1-S G2
ConclusionPluripotent cells: ATR mediates
DNA damage responseDifferentiated cells: ATM
mediates DNA damage response
Differentiated Cells
Pluripotent Cells
Becker, T. and Haferkamp, S., 2013 (Modified by Lucy Lin)
Future StudiesMechanism by which ATR
mediation switches to ATM mediation during differentiation
Examine ATM and ATR inhibition in control SC1 and A-T Q3 iPSCs
AcknowledgmentsRonald P. Hart, Ph.D.Alana Toro-Ramos, Eileen Oni, Angela Tiethof, Tom Pisano, Kunal Garg, Mavis Swerdel, Jennifer Moore, Ph.D.
Lourdes Serrano, Ph.D., Berta Vazquez, Ph.D.
Zhiping Pang, Ph.D., Heather McGowan
Joan Mordes, Program AssistantSURP Directors: Janet Alder, Ph.D., Michael Matise, Ph.D., Mladen-Roko Rasin, M.D., Ph.D.
