DNA damage checkpoints: Mechanisms and Relevance for human cancer ECDO course, Sardinia,September 2006 Jiri Bartek , Danish Cancer Society, Copenhagen
DNA damage checkpoints: Mechanisms andRelevance for human cancer
ECDO course, Sardinia,September 2006
Jiri Bartek, Danish Cancer Society, Copenhagen
S
G1
MMMitosis
G2
Cell cycle control and maintenance of genomic integrity Two layers of control
R
QUIESCENCE(resting state)
DIFFERENTIATION
CELL DEATH
SENESCENCE
PROLIFERATION
Restrictionpoint
S phase
Cell cycle checkpoints
STOP
STOP
STOPSTOP
failure
genetic instability
cancer
uncontrolledcell division
Major cell cycle transitions subverted in cancer
S
M
G1
G2Checkpoints
Restriction pointR Restriction pointRR
S-phase entry
1)
3)Partitioning of
duplicated chromosomes
2)DNA damageCheckpoints
Growth
factors
G0(quiescence)
DNA Damage Replication Fork Arrest
ATM Activation, Relocalization ATR Relocalization
Mediators, Transducers, Effectors
MODULATION OF CELL FATE
[Cell cycle arrests, DNA Repair, Chromatin remodeling, Apoptosis]
Chromosome Instability
CancerKastan M and Bartek J, Nature 432: 316-323, 2004
(if checkpoints fail)
DSB
ATM
Rad50Nbs1
Smc1
Mre11
P
P
G1-Sintra-SG2-M
STOP
rapid;transient
CDC25s
Chk2 Chk1
CyclinsCDKs
PP
P
intra-SSTOP
STOP
STOP
G1-S
G2-M
p53
p21
delayed;sustained
(senescence)STOP
PP
STOP
H2AX
PX
P
CDC25s
Cyclins
Proto-oncogenes
Mdc11
BRCA1
53BP1Mediators
Claspin ATM
Rad50Nbs1
Mre11Chk2
p53
Tumour suppressors
Chk1
BRCA1
p53PP
Mdm2
PP
PP
+MRN
P
Cell cycle impact:
(MRN)P
ATR
Tumour suppressors in DNA damage checkpoint signalling
+ATRIP
Kastan M and Bartek J, Nature 432: 316-323, 2004
Cell deathsubstrates:p53, E2F1, cAbl…..
ATM
Chk2
Cdc25A
Cdc25A
Cdk2Cyc EY15-PT14-
UbCdc25A
Ub
Cdc45 ORI
DNA damage response pathway mediating p53-independent,rapid inhibition of DNA replication and (via Cdk1) G2/M transition
(ATR for UV)
(Chk1 for UV)
Tumour suppressorMutated in: A-T patients
Tumour suppressorMutated in:Colon,lung,breast carcinomas;Li-Fraumeni syndrome
ProtooncogeneOverexpressed in:Breast, lung, head and neckcarcinomas
ProtooncogeneOverexpressed in:Breast, ovarian cancerMailand et al. Science 2000
Falck et al. Nature 2001 Falck et al., Nature Genetics 2002
Mailand et al. EMBO J 2002Sörensen et al. Cancer Cell 2003
βTrc
p1/2
PPPP
DSBS phase
ATR ATM
Chk1
Cdc25A
Basal turnover (T1/2 = 20-30 min)
PP
SCFE2
Ub
Cdc25A
βTrc
p1/2
PPPP
S phase +
ATR
Chk1
Cdc25A
Accelerated proteolysis (T1/2 = 10 min)
PP
SCFE2
Ub
Ub
Cdc25A
Chk2
PP P
S-phase delayS-phase progression
Sørensen et al: Cancer Cell 2003; Syljuåsen et al., Mol. Cell Biol 2005;Bartek J, Lukas C, Lukas J: Nature Rev. Mol. Cell Biol. 2004; Bartek J and Lukas J, Cancer Cell 2003;
The ATR-Chk1 axis as a ‘workhorse’ operating also in normal S phase
DNA Break
ATM Dimer ATM Monomer
Replication Fork Arrest
ATR/ATRIPRPA
ATR/ATRIP (bound to ssDNA)
Claspin
RSR, Rad17
Rad9-Rad1-Hus1 complex
MDC1
53BP1
MRN
BRCA1
Substrate and ATM Relocalization
Substrate Phosphorylation
Cell Cycle Arrest or Apoptosis
Kastan M and Bartek J, Nature 432: 316-323, 2004
DSB
Local chromatin modification
ATMATM
P P
ATM
Autophosphorylationwithin the ATM dimer
Dissociation;conformational change
P
Substrate binding and phosphorylation
S1981-P
ATM
S1981-P
ATM
S1981-P
DSB recession;generation of SS DNA
Coating of SS DNA by RPA
RPA
ATR-ATRIP
RPA-dependent ATR-ATRIP recruitment
ATR
ATRIP
RP
A
P
Substrate phosphorylation
Models of ATM and ATR activation in response to DNA damage
Distinct roles of TopBP1 and claspin in the ATR-mediated DNA damage response
ATRTopBP1
H2AX
Rad17
Nbs1
Smc1
”Kinase X”
ClaspinChk1
DNA damageP
P
P
PPP
P
P ?
P P
Replication stress (UV, HU)Double strand breaks (IR) and replication stress (UV, HU)
TopBP1 dependent, Claspin dependent
TopBP1 dependent, Claspin independent
Claspin is required for Chk1 phosphorylation only
TopBP1 is upstream of Claspin /Chk1, and it is required for phosphorylation of multiple, and probably all, ATR substrates
Liu et al. MCB, August 2006
ATRIP
What dictates the final outcome of the complex cellular DNA damage response, and therefore
decides about cell fate: survival or death?
How are the cell cycle engine (CDKs and proteolysis), cell cycle checkpoints, DNA repair and
cell death/survival pathways coordinated ?
IR-induced ATR activation iscell cycle-dependent
12 16 20 24 28 32Hrs post-release:
pS1981-ATM
ATM
ATR
pT68-Chk2
Chk2
pS317-Chk1
Chk1
Cyclin A
+–IR: +– +– +– +– +–
G1 S G2
Cyclin AGFP-ATR γH2AX
Chk1-pS317 Cyclin A γH2AX
Cyclin ARPA32
Jazayeri et al., Nature Cell Biol., 2006
Proposed model for cell cycle-dependent regulation of DSB repairG1 cells
ATM
Chk2
p53
MRN
No ATRactivation
No resection
Low CDK
G1 arrest
NHEJ
S/G2 cells
ATM
Chk2
RPA-ssDNA
MRN
Rad51/BRCA2
ATR Low CDK(Cell cycle arrest)
High CDK
Chk1
HR
Jazayeri et al. Nature Cell Biol. 2006;
ATM finds NEMOto transiently activate the NF-κB pro-survivalpathway and therebyavoid or delay cell death in response to DNA damage
Is exit from the G2 checkpoint caused by full repair, or adaptation?
IR
DNA damage
G2-arrest
Full repair
Entry into M phase
Adaptation to DNA damage
Entry into M phase
Lethally irradiated cells do exit from the G2 checkpoint:
Clonogenic survival after6 Gy is less than 10%:
1
0.1
Dose (Gy)
0 2 4 6
72 h
48 h
24 h
0 h
DNA content
Cell cycle profiles after 6 Gy: Nuclear fragmentation(6 Gy 72 h):
QuickTime™ and aAnimation decompressor
are needed to see this picture.
After G2 arrest, cells enter M in the presence of γH2AX foci:
Dapi
γH2AX
H3-P
U2OS, mock 6 Gy 40h 6 Gy 40h 6 Gy 40h
Time-lapse video microscopy (U2-OS -GFP-H2B cells):
0 Gy 6 hours after 6 Gy+Chk1-inihibitor
40 hours after 6 Gy
QuickTime™ and aAnimation decompressor
are needed to see this picture.
QuickTime™ and aAnimation decompressor
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QuickTime™ and aAnimation decompressor
are needed to see this picture.
IR
DNA strand breaksChromosome damage
Chk1-dependent G2 arrest
Adaptation
M phase entry
Defective mitosis due to chromosome damage
Nuclear fragmentation / “mitosis-linked death”
Chk1-inhibitor(CEP-3891)
MODEL (timing & adaptation in the G2 checkpoint):
Syljuåsen et al., Cancer Res. 2006
A
DN
A
dam
age
Rep
licat
ion
stre
ssCdc25A
Ub
Chk1Claspin
β-TrcpSCF
Cdc25AP
Cdc25AUb
MITOSIS
P
Plk1 ?
Cdk1Y15-P
Wee1
Xinactive
β-TrcpSCF
Chk1Claspin
Plk1P
ClaspinP
Cdc25A
Chk1
Cdc25ACdc25A
MITOSIS
ClaspinUb
B
Cdc25AP
X
DN
A
dam
age
Rep
licat
ion
stre
ssG
2/M
tran
sitio
n
Wee1P
Wee1Pβ-TrcpSCF
Ub
Wee1Ub
Cdk1Y15-P X
inactive
X
active
β-TRCP-dependent degradation of Claspin limits Chk1-mediated signaling at the G2/M boundary during normal cell cycle progression and during recovery from checkpoint-mediated cell cycle arrest.
Checkpoint adaptation/recovery?
Mailand et al.Mol.Cell, August 2006
DNA damage response and cancer
A) DNA damage causes cancer (through mutations)
B) DNA damage is the major cancer treatment modality (radiotherapy and chemotherapy operate largely through DNA damage)
C) DNA damage is responsible for harmful side-effects of cancer therapy in normal tissues (hair loss, bone marrow and gastrointestinal problems)
D) DNA damage response may serve as a barrier against cancer progression early in human tumour development!
Bartkova et al.
Why do we not alldie of cancer at an early age ?
*Chk2 mutations and activation in human tumours
DiTulio et al.Nature Cell Biol2002
Aberrant constitutive activation of Chk2 in untreated human breast and lung carcinomas
DiTulio et al., Nature Cell Biol.4: 998-1002; 2002.
0
25
50
75
100
Cas
es (
%)
Normal(n=8)
Ta(n=21)
T1(n=25)
T2-4(n=48)
pT-Chk2
0
25
50
75
100
Normal(n=8)
Ta(n=21)
T1(n=25)
T2-4(n=15)
pS-ATM
High Medium Low NegativeNormal Ta T1 T2-4
Chk2
pT-Chk2
ATM
pT-ATM
pS-p53
γ-H2AX
High NegativeMedium Low
0
25
50
75
100
Cas
es (
%)
0
25
50
75
100
Ki67
Constitutive activation of the ATM-Chk2-p53 pathway in early stages of human urinary bladder cancer
Bartkova et al., Nature 2005
γ−H2AX pS-p53 pS-Chk1C
yclin
EC
dc25
AE
2F1
-Te
t+
Tet
-Te
t+
Tet
+ T
am-
Tam
Mcm7
Cdk1 (P-Y15)
days0 2 4 6
cyclin E
+ Tet
1x 2x 4x
Mcm7
RPA
1x 2x 4x
- Tet
cyclin E (- Tet)
Cdk1(total)
pS-ATMγ-H2AX Merge
pT-Chk2γ-H2AX Merge
DNA damage checkpoint response to overexpressed oncogenes in human U-2-OS cells
Flow cytometry profilesafter cyclin E inductionindicate activation of S-
and G2- checkpoints
DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis
• i) The early premalignant lesions (but not the normal tissues) of bladder, breast, lung and colon commonly express markers of activated DNA damage response such as phosphorylated forms of histone H2AX, ATM, Chk2 and p53
• ii) Genetic analysis of human urinary bladder tumours, using a genome-wide assessment of allelic imbalances (via 10.000 SNP arrays) shows that the DNA damage response activation occurs earlier than gross genomic instability.
• iii) Defects in the ATM-Chk2-p53 pathway, such as p53 mutations, occur only after the DNA damage response activation. Hence the activated checkpoints likely create a pressure that selects for p53 and other defects.
• iv) Oncogenic changes that grossly deregulate G1/S transition, such as overexpression of cyclin E, Cdc25A, E2F1, or loss of RB, indeed do induce markers of activated DNA damage in human cultured cells.
• v) Such DNA damage response activation (including the phosphorylated H2AX, ATM, Chk2 and p53) correlated with hallmarks of aberrant DNA replication, including activation of the ATR-Chk1 pathway, enhanced chromatin binding and hyperphosphorylation of RPA, aberrant occurrence of ssDNA regions, and arrest of cells in S and G2 phases, with inactive form of cyclin/CDK complex(es).
PREDICTIONS FOR CLINICAL SETTINGS (early lesions in vivo):• A) High frequency of oncogenic abnormalities that deregulate G1/S control;• B) Evidence for deregulated DNA replication; and• C) Activation of functional cell cycle checkpoint response.
Unscheduled replication
Aberrant replication structures
DNA damage
ATR/Chk1 ATM/Chk2
p53
γ-H2AX
Selection pressureagainst early tumour progression
Growth arrest or cell death
Telomereerosion
Unscheduled replication
Aberrant replication structures
DNA damage
ATR/Chk1 ATM/Chk2
p53
γ-H2AX
Selection pressureagainst early tumour progression
Growth arrest or cell death
Telomereerosion
Activated oncogenes
Model of DNA damage checkpoint activation in response to oncogenic stimuli that deregulate DNA replication (and/or to telomere erosion)
The ATR/ATM-activated network may serve as an inducible barrier to constrain tumour development in itsearly, premalignant stages, and create environment that selects for mutations in checkpoint genes. Tumour-associated defects in the DNA damage response network, such as those in ATM, Chk2 or p53 may rescue defective cell growth and limit cell death at the expense of genomic instability and tumour progression.
(Bartkova et al. Nature, April14, 2005)
T. Halazonetis/V. GorgoulisJ. Bartek
The DNA damage checkpoint is a barrier to tumorigenesis
P. PandolfiD. PeeperC. SchmittM. Serrano
Senescence is a barrier to tumorigenesis
S. Lowe et al., Nature, 2004
DNA damage response activation in early human cancer -Tumour spectrum & Oncogenes
• Lung cancer• Colorectal cancer• Breast cancer• Urinary bladder cancer• Melanoma
(Bartkova et al., and Gorgoulis et al., Nature April 2005)
• Testicular germ cell tumours ?
Clinical implications: Response to therapy (individualized approach) Novel treatment strategies...
Oncogenes: cyclin EE2F1Cdc25A
H-RasMosc-Myc
Cell cycleprogression
Growthfactors
p130
p107Cyclin D1CDK4 Rb E2F
p16
Cyclin ECDK2
Cyclin ACDK2
Emi1
Cdc25A
Cdh1
p27p27
PP
P
P
P
PP
PRb
p16
p27
Tumour suppressors
Cyclin D1CDK4
Emi1
Cdc25A
Cyclin E
Proto-oncogenes
Differential impact of diverse defects within the retinoblastomaprotein pathway on activation of the DNA damage response
*
***
*Subthreshold effects(no DDR activation) * * Suprathreshold effects
(DDR activation)
**
**
**
Defects of DDR genes predispose to familial breast cancer
‘Major’ susceptibility genes: BRCA1BRCA2
‘Minor’ susceptibility genes: ATMChk2p53(PTEN)
These genetic defects together account for some 30-40% of familial BCPrediction: Other DDR factors as potential BC susceptibility genes
How to monitor DSB-induced cell cycle checkpoints in space and time?
γ-H2AX (5 Gy)
Foci...
1 µm
Focused laser beam (λ=337nm)
.
Cells sensitized by BrdU/IdU
Spatially restrictedDNA strand breaks
Laser line
Cell nucleus
γ-H2AX
Lukas C. et al., Nature Cell Biol. 2003Lukas C. et al., EMBO J. 2004Bekker-Jensen et al., JCB, 2005; 2006
Bekker
Why...’in space and time’?
Live cell nucleus
natural conditions for enzymatic reactions
entire spectrum of interaction ’competitors’
physiological local concentrations of interacting proteins
Specific areas(difficult to address
in a test tube...)
Sensing DNA lesions
Processing DNA lesions
Intra-nuclear communication
*How fast?
*Who is first?
*Is the direct contactwith the lesion required?
*Signalosomes orassembly on the spot?
*Processive scanning ordistributive interactions?
*DNA lesion - local chromatin
*DNA lesion - undamaged nucleus
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Real-time imaging of DSBs
Laser path (DSB areas)
0-10 min
Nbs1-YFP
10 min
DSB areas
Nbs1-YFP
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are needed to see this picture.
Rapid recruitment of Nbs1 to DSBs vs. slower Rad52
Nbs1-YFPGFP-Rad52
DSB areas
0-10 min
Spatial patterns of DSB-induced protein redistribution
Chk1, Chk2Chk1
No sustained retention
γ-H2AX Merge
Spreading the signal (from focal DSB to the rest of the nucleus)
T68-PATMChk2
Phospho-Chk2 (Thr68): Global
1 min 5min 10 min 15 min
T68-PChk2Chk2
γ-H2AX
p53Chk2 P
oriCdc25A
Chk2 P
Luciferase
p53-dependent light emission
Luciferase
p53-regulated
Intra-nuclear mobility of Chk2 determines the ’strength’of the p53 tumour suppressor
Immobile(local)
Chk2H2B
Mobile(global)
ActivatedChk2Thr48-P
Chk2
p53-dependent light emission
Chk2
p53Activated after DNA damage
Phosphorylation of p53 at and outside the DSBs
γ-H2AX p53 S15-P
Laser track Laser track
p53
Immobile GFP-p53
GFP
GFP p53 S15-P
Laser track
Laser track
GFP Chk2 T68-P
Chk2
Immobile GFP-Chk2
GFP
H2B
H2B
Chk1
(S31
7-P )
Messengers (focal lesion nucleus)Chk1, Chk2, Kap1
Smc1
(S95
7-P )
Local modifications without recruitmentHistones, Smc1/Smc3 cohesin
53BP
1
DSB-flanking chromatinMdc1, 53BP1, ATM, Mre11, Nbs1, BRCA1
RPA
Processed DNA double strand breaks (DSBs)RPA, ATRIP, ATR, Rad51, Rad52, FancD2,BRCA2, Rad9, Rad17, TopBP1, Nbs1, Mre11,Rad50
γ-H2AX Merge
DNA-P
K
Unprocessed DSBsDNA-PK; Ku70, Ku80
Spatial ‘map’ of nuclear sub-compartments generated by genotoxic stress
Bekker-Jensen et al., J. Cell Biol., 2005 & 2006Ziv et al., Nature Cell Biol. July, 2006 (Kap-1)
Mdc1 triggers structural changes in chromatin micro-compartmentsrequired for a productive assembly of 53BP1
γ−H2AX
H3-dmK79
53BP153BP153BP1
53BP1
Mdc1Mdc1 Mdc1
Allows ’unmasking’ of epigenetic marks otherwisehidden within the compact nucleosomes(recruitment and/or stabilization of chromatin-remodelling factors)
Spatial subclassification of selected proteins involved in DSB response
Erich Nigg
Stephen Jackson
The Wellcome Trust/Cancer Research UKCambridge
Max Planck Institute, Martinsried
Ewa Rajpert DeMeytsNiels SkakkebækMaxwell Sehested
Copenhagen University Hospital
Our external collaborators
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Claudia LukasAlwin KrämerClaus SörensenRandi SyljuåsenFrederic TortAnja Groth
Niels MailandJacob Falck
Fredrik MelanderSimon Bekker-Jensen
Jirina BartkovaZuzana HorejsiJeppe AgnerSanne Jensen
Jiri Lukas
Danish Cancer SocietyCopenhagen
Christopher BakkenistMichael B. Kastan
St. Jude Children’s Research Hospital, memphis
University of Helsinki
Heli Nevanlinna
University of Aarhus
Torben Ørntoft
Per GuldbergMarja Jäättelä
Julio Celis
University of Sheffield
Thomas Helleday
The Sackler Sch. Med., Tel Aviv
Yossi Shiloh