genome maintenance systems, cancer and aging · oliver bischof (pasteur inst), shurong huang (u...
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NIH DNA RepairFeb, 2005
Dr. Judy Campisi
Genome maintenance systems, cancer and aging
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CancerMajor cause of mortality after about the mid-point of maximum life span
Why does it happen?
Why doesn't it happen more often?
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Tumor suppressor mechanisms
curtail the development of
cancer throughout the life span
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Two Classes of Tumor Suppressors
• CARETAKERS -- act on the genome
Prevent cancer by preventing mutationsDamage prevention and repair
• GATEKEEPERS -- act on cellsPrevent potential cancer cells from forming tumors
Apoptosis - causes potential cancer cells to dieCellular senescence - prevents their growth
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Caretaker tumor suppressors arelongevity assurance genes
(e.g., BLM, WRN, TELOMERES)
Gatekeeper tumor suppressors can beantagonistically pleiotropic
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ANTAGONISTIC PLEIOTROPY
(evolutionary hypothesis of aging)
What's good for you when you are young
can be bad for you when you are old.
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Why might gatekeeper tumor suppressors --be antagonistically pleiotropic??
APOPTOSIS -- culls defective cells…..but deplete tissues of cells
CELLULAR SENESCENCE -- arrests proliferation of defective cells …..
but senescent cells are dysfunctional
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Suppressing cancer costs -- aging
Tumor Suppressor mechanisms
AgingPhenotypes
Care-takers(prevent/
repair damage)
Gate-keepers
(eliminate/arrestdamaged cells)
Apoptosis
Senescence
Deplete proliferating/stem cell pools --->
Tissue atrophy/degeneration
Deplete proliferating/stem poolsCell dysfunction ---> loss oftissue function/homeostasis
Late lifephenotypes,
including cancer(antagonisticpleiotropy)
p53
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Bloom Syndrome Werner Syndrome
Life Span = 4-5 decades
Normal until pubertyPremature aging
Cancer prone (mesenchyme)Cardiovascular disease
Type II diabetesCataracts
*
Life Span = 2-3 decades
Small from birthCancer prone (age-type)Sun-sensitive skin rash
Immune-deficientType II diabetesMale infertility
*
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Human RECQ-like helicases
helicaseRecQ1
helicase --> BSBLM
--> WSexo helicaseWRN
helicaseRecQ4/RTS
helicaseRecQ5
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BLM, WRN -- no obvious tissue-specificity
-- no obvious developmental specificity
What are the unique functions of WRN and BLM that might account for
the unique phenotypes of WS and BS?
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BLM PML Merged Dapi
BLM localizes to PML bodies
BLM is expressed in S phase
IP: RAD51 IgG Input (10%)
BLM interacts withRAD51
WB: BLM
WB: RAD51
Role in homologous recombinationG0 G1 G1/S S S S G2
BLM
pro
tein
Bischof et al., J Cell Biol 153: 367 (2001)
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BSF/BLM
BLM
BSF/HM
NHF/HM
NHF
MergePML
NHF
DAPI
NHF
NHF + HM
BSF
BSF + B
BSF + HMLM
Isogenic ‘normal’human BS fibroblasts
+/- BLM Tubulin
BLM
‘Normal’ =telomerized
‘Normal’ =p53 checkpoints
intact
Davalos & Campisi, J Cell Biol 162: 1197 (2003)
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BS cells are mildly hypersensitive to DNA damaging agents:
APOPTOSIS
Also..Bleomycin, X-rays
% A
POPT
OSI
S (M
ito)
10
30
10 30
Etoposide
Wild-typeBSBS + BLMWild-type + HM-BLM
30 30
Com
plem
enta
tion
Dom
inan
t Neg
10 30
WS
Exponentially growing cells
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Damaged BS fibroblasts die by apoptosisNHF BSF + BLMBSF
-
+
HU
0
0.1
0.2
NHF BSF BSF + BLM
untreated
Etoposide
Etoposide +DEVD0.D
. (40
5)
Cells
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Hypersensitivity of BS cells confined to S phaseWild-typeBSBS + BLM
Etoposide, 30 uM
G1 S phase S phase
Wild-type + HM-BLM
% A
popt
osis 60
20
% Labeled Nuclei: <5% >80% >80%
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Hypersensitivity of BS cells in S phasedue to stalled replication forks
Wild-typeBSBS + BLM
Hydroxyurea
G1 S phase S phase
Wild-type + HM-BLM
% A
popt
osis 60
20
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Stalled replication forks develop DSBs;BLM localizes to replication forks with DSBs
Bleomycin HUUntreated
BLM
γ−H2AX
Merge
DAPI
Co-localization: 60-70% >95%
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BLM responds rapidly to damagedreplication forks
% C
ells
with
H2A
X fo
ci
100
50
Time (min) after stall5 15 30 60 %
w C
o-lo
caliz
ed B
LM/H
2AX
IgG
- +
α−BLM
- +
HU
Input (10%)
- +
WB: γ-H2AXIP: BLM
Complex with γ-H2AX
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BS cells undergo apoptosis at high frequency when replication forks stall
Endogenous oxidative damage continuallystalls replication forks
BS cells are continually faced with conditions that favor cell death
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Why are children with BS small?
BS children continually lose cells byapoptosis
(during embryogenesis and throughout life)
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APOPTOSIS SUPPRESSES CANCER
Why are children with BS cancer prone(not progeroid)?
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≈70% BS cells undergo apoptosis whenreplication forks stall
What happens to the survivors?
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Synchronize survivors in S phase
+ HU:
20-30% survival
(Survival is stochastic)
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Apoptotic death of BS cells is p53-dependent%
Apo
ptos
is
50
Cont + E6/DN Cont + E6/DN NF BSF
UntreatedHU
p53
NH
F
BSF
+ 1
43
BSF
BSF
+ E
6
Actin
p53 prevents survival of BS cells withdamaged replication forks
(cells at risk for faulty repair)
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BS cells are under pressure to lose p53function in order to survive damage
during replication
Loss of p53 function + susceptibility to mutations ---->
very high risk for developing cancer
Children with BS die of cancer before high rate of apoptosis can produce
progeroid symptoms
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What is the function of BLM at damagedreplication forks?
What is the role of p53?
Davalos et al., Cell Cycle 3: 1579 (2004)
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BLM
PML
Merge
Dapi
NHF BS/BLM
Untreated
BLM moves transiently from PML bodies to replication forks with DSBs
NHF BS/BLM
HU Stall, 1 h
NHF BS/BLM
HU 1 h + 3 h recovery
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What regulates BLM movement from PML bodies to damaged replication forks?
ATM, ATR damage signaling kinases?
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ATR required for BLM translocation todamaged replication forks
ATR-deficient human Fb (TF) + dox-inducible flag-KD-ATR (dominant negative)Primary NHF, WT or ATR-deficient (Seckle syndrome)
UntreatedATR WT KD
HU, 1 hWT KD
BLM
PML
Merge
Dapi
wt ATR KD ATRDox - - ++
ATR
Actin
PML
ATR Merge
DAPI
PML
ATR Merge
DAPI
WT
ATR
K
D A
TR
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ATM required for BLM return to PML bodiesAT human fibroblasts, +/- telomerase
1 hHU 4 h 6 h
BLM
PML
Merge
Dapi
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Structural function for BLM atdamaged replication forks ?
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BLM required for BRCA1, NBS1 recruitment to damaged replication forks
BRCA1 +/m
BLM PML
Merge DAPI
NBS1 -/-
BLM PML
Merge DAPI
BSF + BLM
NHF
BSF
BRCA1 Dapi NBS1 DAPI
-
+
-
+
-
+
HU
HU - - -+ + +NHF BSF BSF+BLM
BRCA1Actin
BLM
NBS1
Input (10%) IP: IgG IP: BLMHU - + - + - + WB:
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Catalytically inactive BLM restores BRCA1, NBSIlocalization to damaged replication forks
BRCA1 DAPI NBS1 HU DAPI
--
NHF
++
--
BSF
++
NBS1 BRCA1 DAPI Merge
--
BSF + HM ++
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BLM required for 53BP1 recruitment and ATM activation
BS
F1
Unt
Bleo
HU
ATM S1981 53BP1 Merge N
HF
Unt
Bleo
HU
ATM S1981 53BP1 Merge
- +
53BP1
NHF
- +
BSF/BLM
- +
BSF NHF
HUHU - 1h 4h
ATM S1981ATM S1981
ATM Actin
Actin
Actin
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What is the role of p53 in BLM function?
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p53 prevents repair complexes from formingin absence of BLM
0
20
40
60
80
100
UntHU
FociP53 status
BRCA1 BRCA1 NBS1 NBS1WT Mut WT Mut
% C
ells
with
foci
NBS1
BRCA1
NHF + E6 + HUBS Cells -- 1 h after stall
HU - + ATM S1981 53BP1 Merge
ATM S1981 BSF+
DN-p53
ATM
Actin
53BP1
Actin
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p53 orchestrates assembly of repair complexesat replication forks with DSBs(prevent error-prone repair?)
BLM is crucial for orchestrated,p53-dependent repair complex assembly
BLM + p53 deficiency ---> genomic instability,(cancer)
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Werner Syndrome
Life Span = 4-5 decades
Normal until pubertyPremature aging
Cancer prone (mesenchyme)Cardiovascular disease
Type II diabetesCataracts
*
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Human RECQ-like helicases
helicaseRecQ1
helicaseBLM
WRN exo helicase
helicaseRecQ4/RTS
helicaseRecQ5
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WRN is not cell cycle regulated;Localizes to nucleoli
N le
vel
WR
G0 G1 S G2 M
WRN B23 Merge DAPI
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WRN interacts with components of NHEJ
Yannone et al., J Biol Chem 276: 38242 (2001)
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WS 5' d
elet
ion
BR
EAK
3' d
elet
ion
WRN deficiency causes extensive 3' deletion at non-homologously joined ends
Oshima et al., Cancer Res 62: 574 (2002)
WS + WRNWild-type
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WRN can interact with NHEJ components and participates in NHEJ reactions
NHEJ requires a helicase to search for microhomology and exonuclease to
process broken ends
Cells defective in NHEJ are extremelysensitive to IR
WS cells are mildly IR-sensitive
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WS cells are mildly X-ray sensitive
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Under what circumstances is the WRN-NHEJ interaction relevant?
Dysfunctional telomeres resemble DNADSBs, substrates for NHEJ
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WRN deficiency accelerates senescence(in contrast to BLM deficiency, which increases apoptosis)
(premature accumulation of senescent cells -->loss of tissue function & homeostasis,
promotes late life cancer)
Abrogated by telomerase (epithelial stem cells)
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Thanks!
Present Lab members: Albert Davalos,Seok-Jin Heo, Patrick Kaminker, Francis Rodier
Past Lab members: Junko Oshima (UWA)Oliver Bischof (Pasteur Inst),
Shurong Huang (U WA), Sashwati Roy (OSU)
Collaborators: Sandy Chang (MDAnderson),David Chen/Steve Yannone (LBNL),
Nathan Ellis (Sloan-Kettering), Junko Oshima(U WA), Robert Shiestl (UCLA)