The role of Base Excision Repair genes
in DNA damage response induced by
chemotherapeutic drugs
Cláudio Pinheiro
Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology
(ToxOmics)
NOVA Medical School/Faculdade de Ciências Médicas
E-mail: [email protected]
Introduction
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
• The cells of our body are permanently
exposed to reactive agents capable of
damaging the cellular components;
• DNA can accumulate damage over a
lifetime;
• DNA is the only biological molecule that
relies solely on repair of existing
molecules, without any resynthesizes.
Introduction
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
• Organisms have evolved rapid and efficient mechanisms of DNA repair
Adapted from Hoeijmakers 2001
Introduction
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
Silenced genes
(HeLa SilenciX®)
•
AP endonuclease (APE1)
–
•
Poly(ADP-ribose) polymerase 1 (PARP1)
–
•
Creates a nick in the phosphodiester
backbone of the AP site
Modifies nuclear proteins by
poly ADP-ribosylation
X-ray cross complementing protein 1
(XRCC1)
–
Recruits enzymes that will carry out
their enzymatic roles in repairing the
DNA
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
Introduction
Doxorubicin:
5-fluorouracil
(Anthracicline antitumor antibiotic)
(Anti-metabolite)
DOX binds at the
topoisomerase II
interface, which
promotes the
formation of
covalent cleavage
complexes.
DOX is also
a redox
active
compound
5-FU incorporated
into the cells is
converted into
active metabolites
capable of acting as
pyrimidine
analogous
Misincorporated
into both DNA
and RNA
Generates
double
stranded
breaks
Increases the
levels of
Reactive Oxygen
Species (ROS)
Lethal
DAMAGE
Paclitaxel
(Taxane)
Irreversible
inhibition of the
thymidilate
synthase
Catalyse the
conversion of
deoxyuridine
monophosphate
(dUMP) to
deoxythymidine
monophosphate
(dTMP)
Arresting the
cell culture in
the S Phase
Paclitaxel is a
taxane compound
that binds to the βtubulin stabilizing it.
Stabilization of microtubules
prevents normal formation of
mitotic spindles.
The lack of microtubules dynamics
prevents the chromosome biorientation.
Mitotic arrest
Comet assay
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
Single Cell Gel Electrophoresis assay
A sensitive technique for the detection of DNA
damage at the level of the individual eukaryotic cell
Alkaline Treatment
Negative Control (NC)
No exposure
Allows the detection
of DNA strand breaks
(SBs) and alkali-labile
sites (ALS)
Positive Control (PC)
10 µM of H2O2
Comet assay
•
•
•
•
•
NC
PC
Dox 500nM
Pax 1nM
5-FU 200µM
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
Exposure time:
60 min
Recovery time points:
15 min
30 min
60 min
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
Results
0 min
NC
100
% DNA in tail
WT
WT
APEXPARP1XRCC1-
80
60
15 min
40
20
30 min
0
0
15
30
45
60
Recovery time (min)
60 min
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
Results
0 min
PC
†
% DNA in tail
100
WT
†
WT
APEX-
80
PARP1-
60
15 min
-
XRCC1
40
20
30 min
0
0
15
30
45
60
Recovery time (min)
60 min
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
Results
0 min
DOX
100
WT
APEX-
80
% DNA in tail
WT
PARP1-
60
15 min
-
XRCC1
40
20
30 min
0
0
15
30
45
60
Recovery time (min)
60 min
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
Results
0 min
5-FU
100
WT
APEX-
80
% DNA in tail
WT
PARP1-
60
15 min
-
XRCC1
40
20
30 min
0
0
15
30
45
60
Recovery time (min)
60 min
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
Results
0 min
PAX
100
WT
APEX-
80
% DNA in tail
WT
PARP1-
60
-
15 min
XRCC1
40
20
30 min
0
0
15
30
45
60
Recovery time (min)
60 min
Conclusions
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
• All the cell lines recovered from the damage caused by the
chemotherapeutic agents.
• APEX- and XRCC1- cell lines were the most sensitive when
exposed to the agents.
• The silenced proteins do not affect the resilience of the cells,
mostly due to the action of complementary mechanisms of
repair that act simultaneously to BER pathway.
• Our results lead to some further questions, such as, the
relevance of apoptosis pathways or other repair mechanisms
(e.g. HR)
Acknowledgments
The role of Base Excision Repair genes in DNA damage
response induced by chemotherapeutic drugs
Professor Doutor José Rueff
Doutora Susana Nunes da Silva
Doutora Helena Borba
SFRH/BPD/ 80462/2011
E-mails: [email protected]
[email protected]
UID/BIM/00009/2013