NIH DNA RepairFeb, 2005

Dr. Judy Campisi

Genome maintenance systems, cancer and aging

CancerMajor cause of mortality after about the mid-point of maximum life span

Why does it happen?

Why doesn't it happen more often?

Tumor suppressor mechanisms

curtail the development of

cancer throughout the life span

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

Caretaker tumor suppressors arelongevity assurance genes

(e.g., BLM, WRN, TELOMERES)

Gatekeeper tumor suppressors can beantagonistically pleiotropic

ANTAGONISTIC PLEIOTROPY

(evolutionary hypothesis of aging)

What's good for you when you are young

can be bad for you when you are old.

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

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

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

*

Human RECQ-like helicases

helicaseRecQ1

helicase --> BSBLM

--> WSexo helicaseWRN

helicaseRecQ4/RTS

helicaseRecQ5

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?

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)

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)

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

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

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%

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

Stalled replication forks develop DSBs;BLM localizes to replication forks with DSBs

Bleomycin HUUntreated

BLM

γ−H2AX

Merge

DAPI

Co-localization: 60-70% >95%

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

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

Why are children with BS small?

BS children continually lose cells byapoptosis

(during embryogenesis and throughout life)

APOPTOSIS SUPPRESSES CANCER

Why are children with BS cancer prone(not progeroid)?

≈70% BS cells undergo apoptosis whenreplication forks stall

What happens to the survivors?

Synchronize survivors in S phase

+ HU:

20-30% survival

(Survival is stochastic)

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)

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

What is the function of BLM at damagedreplication forks?

What is the role of p53?

Davalos et al., Cell Cycle 3: 1579 (2004)

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

What regulates BLM movement from PML bodies to damaged replication forks?

ATM, ATR damage signaling kinases?

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

ATM required for BLM return to PML bodiesAT human fibroblasts, +/- telomerase

1 hHU 4 h 6 h

BLM

PML

Merge

Dapi

Structural function for BLM atdamaged replication forks ?

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:

Catalytically inactive BLM restores BRCA1, NBSIlocalization to damaged replication forks

BRCA1 DAPI NBS1 HU DAPI

--

NHF

++

--

BSF

++

NBS1 BRCA1 DAPI Merge

--

BSF + HM ++

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

What is the role of p53 in BLM function?

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

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)

Werner Syndrome

Life Span = 4-5 decades

Normal until pubertyPremature aging

Cancer prone (mesenchyme)Cardiovascular disease

Type II diabetesCataracts

*

Human RECQ-like helicases

helicaseRecQ1

helicaseBLM

WRN exo helicase

helicaseRecQ4/RTS

helicaseRecQ5

WRN is not cell cycle regulated;Localizes to nucleoli

N le

vel

WR

G0 G1 S G2 M

WRN B23 Merge DAPI

WRN interacts with components of NHEJ

Yannone et al., J Biol Chem 276: 38242 (2001)

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

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

WS cells are mildly X-ray sensitive

Under what circumstances is the WRN-NHEJ interaction relevant?

Dysfunctional telomeres resemble DNADSBs, substrates for NHEJ

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)

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)