...................................................................................................................................
3 TABLE
OF
CONTENT
.....................................................................................................................
4 1
INTRODUCTION
........................................................................................................................
7 1.1
GENERAL
INTRODUCTION
.................................................................................................................
7 1.2
THE
DNA
DAMAGE
AND
REPLICATION
STRESS
RESPONSE
........................................................................
8 1.2.1
DNA
damage
response
......................................................................................................
8 1.2.2
Replication
stress
...............................................................................................................
9 1.2.3
DDR
kinases
.....................................................................................................................
10 1.2.4
Phosphatases
...................................................................................................................
16 1.2.5
DNA
damage
induced
cell
cycle
checkpoints
...................................................................
16 1.2.7
DNA
repair
.......................................................................................................................
18 1.4
HYPOXIA
....................................................................................................................................
19 1.4.1
Hypoxia
in
human
tumors
...............................................................................................
19 1.4.2
Hypoxia-‐induced
genomic
instability
and
replication
stress
............................................
20 1.4.3
Targeting
hypoxic
cells
for
cancer
therapy
......................................................................
21 1.4.4
Desferrioxamine
(DFO)
as
a
hypoxia-‐mimetic
agent
.......................................................
22 2
AIM
........................................................................................................................................
23 3
MATERIALS
.............................................................................................................................
25 3.1
CELL
CULTURE
.............................................................................................................................
25 3.2
SIRNA
TRANSFECTION
..................................................................................................................
25 3.3
FLOW
CYTOMETRY
.......................................................................................................................
26 3.4
IMMUNOFLUORESCENCE
MICROSCOPY
.............................................................................................
26 3.5
SDS-‐PAGE
AND
WESTERN
BLOT
....................................................................................................
27 3.6
CLONOGENIC
SURVIVAL
ASSAY
........................................................................................................
28 3.7
BUFFERS
AND
SOLUTIONS
..............................................................................................................
29 4
METHODS
...............................................................................................................................
31 4.1
CELL
CULTURE
AND
CELL
SEEDING
....................................................................................................
31 4.2
WEE1-‐
AND
ATR-‐INHIBITION
........................................................................................................
32 4.3
SIRNA
TRANSFECTION
..................................................................................................................
32 4.4
HYPOXIA
TREATMENTS
..................................................................................................................
33 4.5
FLOW
CYTOMETRY
.......................................................................................................................
33 4.6
IMMUNOFLUORESCENCE
MICROSCOPY
.............................................................................................
36 4.7
SDS-‐PAGE
................................................................................................................................
37 4.8
WESTERN
BLOT
...........................................................................................................................
37 4.9
CLONOGENIC
SURVIVAL
ASSAY
........................................................................................................
38 5
RESULTS
.................................................................................................................................
39 5.1
VALIDATE
CANDIDATE
HITS
WITH
SIRNA
SCREEN
................................................................................
39 5.2
DETERMINE
CONCENTRATION
OF
ATR-‐INHIBITOR
VE822
....................................................................
42 5 5.3
EFFECTS
OF
MK1775
AND
VE822
TREATMENT
IN
COMBINATION
WITH
HYPOXIA
.....................................
43 5.4
EFFECTS
OF
MK1775
AND
VE822
TREATMENT
AFTER
HYPOXIA
............................................................
46 5.5
MEASUREMENT
OF
CELL
SURVIVAL
...................................................................................................
51 5.6
FLUORESCENCE
IMAGING
OF
MK1775
AND
VE822
TREATED
CELLS
.......................................................
52 5.7
EXPLORING
THE
MECHANISM
BEHIND
THE
SYNERGISTIC
EFFECT
BETWEEN
MK1775
AND
VE822
.................
54 5.8
DESFERRIOXAMINE
AS
A
REPLACEMENT
FOR
HYPOXIA
CHAMBER
.............................................................
59 6
DISCUSSION
............................................................................................................................
61 6.1
GENERAL
DISCUSSION
...................................................................................................................
61 6.2
VALIDATION
OF
SIRNA
SCREEN
.......................................................................................................
61 6.3
COMBINED
WEE1
AND
ATR
INHIBITION
LEADS
TO
SYNERGISTIC
INCREASE
OF
S-‐PHASE
DNA
DAMAGE.
.........
63 6.4
DO
BOTH
WEE1
AND
ATR
INHIBITION
LEAD
TO
ELEVATED
CDK
ACTIVITY?
..............................................
63 6.5
COMBINATION
TREATMENT
WITH
MK1775
AND
VE822
AS
A
POTENTIAL
ANTI-‐CANCER
STRATEGY
...............
64 6.6
MICRONUCLEI
IN
RESPONSE
TO
ATR
INHIBITION
.................................................................................
65 6.7
EXPERIMENTAL
CONSIDERATION
......................................................................................................
65 6.7.1
Cell
culture
.......................................................................................................................
65 6.7.2
siRNA
transfection
...........................................................................................................
66 6.7.3
WEE1
and
ATR
inhibition
.................................................................................................
67 6.7.4
Hypoxia
treatments
.........................................................................................................
67 6.7.5
Measuring
protein
levels
by
flow
cytometry
...................................................................
68 6.7.6
Measuring
protein
levels
by
western
blotting
.................................................................
69 6.8
CONCLUDING
REMARKS
.................................................................................................................
70 7
SUPPLEMENT
..........................................................................................................................
71 8
ACKNOWLEDGEMENTS
...........................................................................................................
73 9
LIST
OF
ABBREVIATIONS
.........................................................................................................
75 10
REFERENCES
.........................................................................................................................
79