Download ppt - Studio dei meccanismi di riparazione del danno ossidativo: modelli “in vitro“ e “in vivo”

Studio dei meccanismi di Studio dei meccanismi di riparazione del danno riparazione del danno

ossidativo: modelli “in vitro“ e ossidativo: modelli “in vitro“ e “in vivo”“in vivo”

11 marzo 2005Centro Ricerche ENEA Casaccia

M. Bignami M. Bignami Istituto Superiore di Sanita’Istituto Superiore di Sanita’

abasic site

NNNNNH2OHOHHHHCHHOH

(5(5'S)-'S)-8,58,5'-cyclo'-cyclo-2-2''-deoxyadenosine-deoxyadenosine

Reactive Oxygen SpeciesReactive Oxygen Species

8-OH-Adenine

8-oxoguanine8-oxoguanine

2-OH-Adenine2-OH-Adenine

2-OH-Ade :G2-OH-Ade :G2-OH-Ade :T2-OH-Ade :T

GC -GC ->AT>ATGC -GC ->TA>TA

AT -AT ->CG>CGAT -AT ->GC>GCAT -AT ->TA>TA

H. Kamiya NAR 31: 517, 2003

GC -> TAGC -> TAAT -> CGAT -> CG

8-oxoG : C8-oxoG : Canti:antianti:anti

8-oxoG : A8-oxoG : Asyn:antisyn:anti

Cheng 1992 J.Biol. Chem267: 166

Base ExcisionBase ExcisionRepairRepair

NucleotideNucleotideExcisionExcisionRepairRepair

MutT hydrolases Mismatch Repair

Proofreading by polymerases

Polymerase selectivity

Translesion synthesis

Pre-replication Replication Post-replication

5’

5’

5’

3’

Post-replicativePost-replicative

G*G*MYHMYH

AA A

G*G*CC

OGG1OGG1

G*G*

CCG*G*

ROS Pre-replicativePre-replicative

CC

Removal of 8-oxoG by Base Excision RepairRemoval of 8-oxoG by Base Excision Repair

+ long patch BER

NucleotideNucleotideExcisionExcisionRepairRepair

•CSB-/- MEFs are hyper-CSB-/- MEFs are hyper-sensitive to sensitive to -irradiation-irradiation•CSB -/- exposed to DHEP CSB -/- exposed to DHEP show weight reductionshow weight reductionDe Waard MCB 2004, 24: 7941

•Primary fibroblasts of CS patients are defective in repair of 8-oxoGTuo et al., FASEB, 2003 17:668

•CSB gene product is involved in general genome BER of 8-oxoGTuo et al., J.B.C., 2001 276:45772

•XPC-HR23B interacts XPC-HR23B interacts with with DNA glycosylases DNA glycosylases involvedinvolved in BER of endogenous in BER of endogenous lesions (lesions (thymine DNA glycosylase, 3-methyl-adenine-DNA- glycosylase)

Cleansing of the oxidized dNTP pool by MutT Cleansing of the oxidized dNTP pool by MutT homologueshomologues

A*A*G/C/AG/C/A

AA

G*G*

Fujikawa et al., 1999 J.Biol. Chem. 274:18201

ROS

8-oxo-dGTP

8-oxo-dGDP

8-oxo-dGMP

DNA

MTH1MTH1

NUDT5NUDT5

Ishibashi T, et al. EMBO Rep. 2003, 4:479

2-OH-dATP

2-OH-dAMP

MTH1MTH1

MismatchMismatch formationformation

Mismatch Mismatch removalremoval

DNA DNA resynthesisresynthesis

MutLMutL: MLH1/PMS2: MLH1/PMS2MutLMutL: MLH1/PMS1: MLH1/PMS1

PolPol//PCNAPCNARPARPARFCRFC

ExonucleaseExonuclease

MismatchMismatch bindingbinding

MutSMutS: MSH2/MSH6: MSH2/MSH6

MutSMutS: MSH2/MSH3: MSH2/MSH3

Does mismatch repair recognize 8-oxoG-containing Does mismatch repair recognize 8-oxoG-containing mismatches?mismatches?

• MSH2 and MSH6 are required for removal of adenine misincorporated opposite 8-MSH2 and MSH6 are required for removal of adenine misincorporated opposite 8-oxoG oxoG (Mol Cell. 1999;4:439)(Mol Cell. 1999;4:439)

• In the absence of In the absence of MSH2/MSH6MSH2/MSH6 mutation rates are highly elevated. When cells mutation rates are highly elevated. When cells are grown anaerobically, the rates are decreased (PNAS 1998, 95:15487)are grown anaerobically, the rates are decreased (PNAS 1998, 95:15487)

hMLH1- and hMLH1-/cDNA human tumor

cells

msh2msh2+/++/+ andand msh2msh2-/--/- MEFs MEFs

Paolo Degan, IST, GenovaPaolo Degan, IST, Genova

2

4

6

8

8-o

xodG

/ 10

6 d

G

2

6

10

8-o

xoG

/10

6 d

G

H2O2-induced levels levels

DNA 8-oxoGDNA 8-oxoG accumulates in DNA of MMR-defective accumulates in DNA of MMR-defective cellscells

msh2msh2-/--/-

0,4

0,8

1,2

1,6

8-o

xodG

/ 10

6 d

G

A2

78

0

+ c

DN

A

0,2

0,4

0,6

0,8

8-o

xoG

/10

6 d

G

+/+/++

-/--/-+/-+/-

Steady-state Steady-state levelslevels

hMLH1-

““in vitro” BER of 8-in vitro” BER of 8-oxoG is unaffected by oxoG is unaffected by the absence of MMR the absence of MMR

proteinsproteins

Extracts from Extracts from msh2msh2+/++/+ and and msh2msh2-/--/-

MEFsMEFs3232P-dNTPsP-dNTPs

8-oxoG8-oxoG5’

3’

in in vitrovitro

synthesisynthesiss

multiplemultiplecloning sitescloning sites

RE digestions RE digestions and and gel gel electrophoresiselectrophoresis

Repair assayRepair assay

extracts from extracts from msh2msh2+/++/+

and and msh2msh2-/--/- MEFs MEFs

3232PP5’

5’

3’3

’

8-oxoG8-oxoGC

Incision assayIncision assay

The The accumulatioaccumulation of 8-oxoG n of 8-oxoG

in MMR-in MMR-defective defective

cells cannot cells cannot be be

accounted accounted for by a for by a

differential differential efficiency of efficiency of

BERBER

msh2/ogg1

-/-

Steady-stateSteady-state

8-o

xodG

resi

dues/

10

6 d

G

0,2

0,6

1

1,4

1,8

ogg1

+/+ -/-+/+-/-

msh2

Msh2 and Ogg1 act independently and Msh2 and Ogg1 act independently and their effects are additivetheir effects are additive

Colussi C, et al. Current Biology, 11:912 Colussi C, et al. Current Biology, 11:912 20022002

msh2-/- ogg1-/-

x

msh2-/- ogg1-/-

MEFs

overexpressionoverexpression of MTH1of MTH1

decreased decreased DNA 8-oxoGDNA 8-oxoG

in MMR-in MMR-defective defective

cells cells

?? decreased ?? decreased mutagenesis mutagenesis

8-o

xod

G /

10

6 d

G

0

0,2

0,4

0,6

0,8

1

+/+ -/-

MTH1 MTH1 activityactivity 1--6 1--10--50

Colussi C, et al., Current Biology, 11:912 2002Colussi C, et al., Current Biology, 11:912 2002

Pre-replication Replication Post-replication

5’

5’

5’

3’

MutT hydrolases

Mismatch Repair

The mutator phenotype of The mutator phenotype of msh2msh2-/--/- MEFs is MEFs is almost abolished by hMTH1 overexpressionalmost abolished by hMTH1 overexpression

hMTH1 hMTH1 (U/mgprotein)(U/mgprotein)

0

20

40

60m

uta

tion r

ate

s x 1

0m

uta

tion r

ate

s x 1

0-7-7

Msh2Msh2-/--/-

Mean: 3,1 x 10-6

1°

2°

3°

0,40,4

Clone 2

3,93,9

0

1

2

3

Clone 5

Clone 5

Mean: 1,8 x 10-7

2020

HPRTHPRT gene gene

hMTH1 overexpression decreases all hMTH1 overexpression decreases all mutational classesmutational classes

muta

tion r

ate

x 1

0m

uta

tion r

ate

x 1

0-7

-7

/cell/

genera

tion

/cell/

genera

tion

TransversionsTransversionsFrameshiftsFrameshifts

TransitionsTransitions

msh2msh2-/--/-

00

44

88

1212

00

44

88

1212

msh2msh2-/--/- ++hMTH1hMTH1

5’3’ CCCCCC G3’AAGGGGGGC

AGGGGGGTTCCCCCCG

5’3’

5’

-1 frameshifts-1 frameshifts

AT>GCAT>GC 24%24% 44.1 x44.1 x

AT>TAAT>TA 12% 12% 61.7 x61.7 x

Transitions

Transversions

AT>CGAT>CG 6% 6% 3.2 x3.2 x

C:C:A*A*2-oxodA

GC>TAGC>TA 6% 6% 32 x32 x

GC>CGGC>CG 6% 6% 11 x11 x

2-OHdATP and 8-oxodGTP can account for a 2-OHdATP and 8-oxodGTP can account for a substantial fraction of the mutator phenotype of a substantial fraction of the mutator phenotype of a

MMR-defective cellMMR-defective cell

2-oxodA

A:A:A*A*

A:A:G*G*8-oxodG

8-oxodG G*:G*:AA

G: G: A*A*2-oxodA8-oxodG G*:G*:GG

MMR is an important protection against MMR is an important protection against spontaneous mutation and human spontaneous mutation and human

cancercancer

hMLH1hMLH1 HypermethylatioHypermethylatio

nn

Mutations in Mutations in hMSH2hMSH2 or or hMLH1hMLH1

SPORADISPORADICC

FAMILIAL FAMILIAL (HNPCC)(HNPCC)

Colorectal Colorectal CancerCancer

Loss of Loss of MMRMMR

MicrosatelliteMicrosatelliteInstabilityInstability

in vitroin vitro100-fold increase100-fold increasein mutation ratesin mutation rates

Mutator phenotypeMutator phenotype

Overexpression of hMTH1 reduces microsatellite Overexpression of hMTH1 reduces microsatellite instability of human MMR-defective cell linesinstability of human MMR-defective cell lines

Muta

tion r

ate

x 1

0M

uta

tion r

ate

x 1

0-3-3

12

8

0

4

DLD1DLD1

DLD1DLD1+MTH1+MTH1

BAT26 (A26)BAT26 (A26)

0

10

20

Muta

tion r

ate

x 1

0M

uta

tion r

ate

x 1

0-3-3

DU145DU145

DU145DU145+MTH1+MTH1

SMT15 (G15)SMT15 (G15)

An important component of MSI at mononucleotide An important component of MSI at mononucleotide AA and and GG

runs depends on incorporation of oxidized precursorsruns depends on incorporation of oxidized precursors

GCATT GCATT GGGGGGGGGGGGGGGG ACACACAC

ATGCGGTAT ATGCGGTAT AAAAAAAAAAAAAAAAAA(26)(26) CTGATGCA CTGATGCA

ROS

MTH1MTH1

Oxidized Oxidized dNTPdNTP

Incorporation Incorporation of oxidized of oxidized purines into purines into

DNA DNA contributes contributes significantly significantly

to the to the genome genome

instability of instability of MMR- MMR-

deficient deficient cellscells

Russo MT, Blasi MF, Chiera F, Fortini P, Degan P, Macpherson P, Furuichi M, Nakabeppu Y, Karran P, Aquilina G, Bignami M. Mol. Cell. Biol. 2004, 24:465-74.

8-OH-dGTP8-OH-dGTP2-OH-dATP2-OH-dATP

8-OH-8-OH-dGMPdGMP2-OH-2-OH-dAMPdAMP replicationreplication

errorserrors++

oxidative oxidative DNA DNA

damagedamage

““Mutator Mutator phenotype”phenotype”

A G G G G G G CA G G G G G G C

C C C GC C C G

G G G

T C C C C C C GT C C C C C C G

8-8-oxoGoxoG

5’

3’

Mouse HPRTMouse HPRT-1 frameshifts-1 frameshifts

5’3’ T CCCCCG

3’5’ AGGGGGC*

C

How easy is it to incorporate and elongate an 8-oxodGTP?

Where do the frameshift occur?

Frameshifts in runs of A and G-from which oxidized triphosphates?

√

√

√

IncorporatioIncorporationand nand

elongation elongation of 8-of 8-

oxodGTPoxodGTPopposite Copposite C

aactcgtgtgtctcc5’

5’agaacttatag ccccccc ttgagcacacagagg3’

primer16-mer

15-mer

17-mer18-mer

19-mer20-mer

21-mer

dGTP (M)dNTP(M)

8-oxodGTP (M) -- -0.1 0.4 1.2 3.80.1 0.4 1.2 3.8

- - - + - + - -+ + +

0.04 - - - - - - - - - 0.04

Klenow Klenow polymerasepolymerase

39-mer

8-oxo8-oxodGTPdGTP

F. BaroneF. Barone

IncorporatioIncorporation and n and

elongation elongation of 8-of 8-

oxodGTPoxodGTPopposite Aopposite A

aactcgtgtgtctcc5’

5’agaacttatag aaaaaa ttgagcacacagagg3’

dGTP (M)

dNTP (M)

- - 12-

- + - +

- - 375

-

375 -

-

-

+

-

-

40

-

+

120

-

-

-

+

375

-

-

-

+

8-oxodGTP (nM)


primer16-mer15-mer

39-mer

F. BaroneF. Barone

aactcgtgtgtctcc5’

5’ ccccccc ttgagcacacagagg3’

dGTP (M) +-- + - + 10- + -

- 3 10 30 -3 10 30 100 100

IncorporatioIncorporationn

and and elongation elongation

of 8-of 8-oxodGTP oxodGTP

opposite Copposite C

human human polymerase polymerase

8-oxodGTP (M)

primer 15-mer

F. BaroneF. BaroneG. MagaG. Maga

dGTP (M) --- -10 - - -

- 3 10 30- 3 10 30

10

-

--

11

dTTP (M) - - - - - -

-

-

10 10 10 10 10 10

aactcgtgtgtctcc5’

5’ aaaaaa ttgagcacacagagg3’

IncorporatioIncorporation of 8-n of 8-

oxodGTP oxodGTP opposite Aopposite A

No No elongationelongation


8-oxodGTP (M)

primer 15-mer16-mer

F. BaroneF. BaroneG. MagaG. Maga



Polymerase Polymerase familyfamily

AA

BB

DPO4DPO4YY

CCCCCCInc. Elong.

++

++

--

++

++

--

++

++

++

++

--

--

AAAAAAInc. Elong.

polpol

polpol

polpolpolpol

Depending on the polymerase 8-oxodGTP be incorporated and elongated in C or A runs

2-fold 2-fold (liver)(liver)


2-fold (MEFs)

2-fold (MEFs)2-fold (MEFs)

cancercancer

_

_

liver, lung,liver, lung, stomachstomach

lymphomaslymphomasGI tractGI tract

Ogg1Ogg1-/--/-

MyhMyh-/--/-

MthMth-/--/-

Msh2Msh2-/--/-

??

??

Hereditary Non Polyposis Colon

CancerLeukemia, lymphoma

biallelic mutations in Familial

Adenomatous Polyposis (FAP)

Accumulation of 8-oxoG and cancer ?Accumulation of 8-oxoG and cancer ?

WT

liverliver

0

0,4

0,8

1,2

1,6

2

4 8 120

0,4

0,8

1,2

1,6

2

4 8 12

8-o

xodG

resi

dues/

10

6 d

G

0,4

0,8

1,2

1,6

2

0 4 8 12

small intestinesmall intestine lunglung

In wild-type animalsIn wild-type animals steady-state steady-state levels of DNA 8-oxoG do not vary levels of DNA 8-oxoG do not vary

with agewith age

kidneykidney

2

0,4

0,8

1,2

1,6

0 4 8 12 0

0,4

0,8

1,2

1,6

2

4 8 12

0,4

0,8

1,2

1,6

2

0 4 8 12

brainbrain spleenspleen

months months months

8-o

xodG

resi

dues/

10

6 d

G

Osterod et al, Carcinogenesis 2001

Levels of 8-oxoG increase in BER-defective Levels of 8-oxoG increase in BER-defective mice: mice: liverliver

Post-replicativePost-replicative

G*G*

MYHMYH

AA A

OGG1OGG1

G*G*

CC

G*G*

ROS

Pre-replicativePre-replicative

months

0,4

0,8

1,2

1,6

2

0 4 8 12 16

Ogg1-/-

Myh-/-

0,4

0,8

1,2

1,6

2

0 4 8 12 16

Myh-/-

8-o

xod

G r

esi

du

es/

10

6 d

G

months

Ogg1-/-

Osterod et al, Carcinogenesis 2001

Levels of 8-Levels of 8-oxoG oxoG

increase increase synergisticallsynergisticall

y in BER-y in BER-defectivedefective

mice

Russo MT, De Luca G, Degan P, Parlanti E, Dogliotti E, Barnes DE, Lindahl T, Yang H, Miller JH, Bignami M. Cancer Res. 2004 Jul

1;64:4411-4.

0

0,4

0,8

1,2

1,6

2

4 8 12 16

months

months

0,4

0,8

1,2

1,6

2

0 4 8 12 16

Myh-/-

8-o

xod

G r

esi

du

es/

10

6 d

G8

-oxo

dG

resi

du

es/

10

6 d

G

lung

Small intestine

months

0

0,4

0,8

1,2

1,6

2

4 8 12 16

0,4

0,8

1,2

1,6

2

0 4 8 12 16

months

Ogg1-/-

Myh-/-

BrainBrain

KidneyKidney

SpleeSpleenn

months

0,4

0,8

1,2

1,6

2

0 4 8 12 16

0,4

0,8

1,2

1,6

2

0 4 8 12

WTWT

8-o

xod

G r

esi

du

es/

10

6 d

G

2

0,4

0,8

1,2

1,6

0 4 8 12

0

0,4

0,8

1,2

1,6

2

4 8 12

0,4

0,8

1,2

1,6

2

0 4 8 12 16

0,4

0,8

1,2

1,6

2

0 4 8 12 16

MyhMyhOgg1Ogg1

0 4 8 12 16

0,4

0,8

1,2

1,6

2

2

0,4

0,8

1,2

1,6

0 4 8 12 16

0,4

0,8

1,2

1,6

2

0 4 8 12 16

MyhMyh

No accumulation in the levels of DNA 8-oxoG No accumulation in the levels of DNA 8-oxoG was observed in other organs of was observed in other organs of MyhMyh-/--/-Ogg1Ogg1-/--/-

micemice

This is the only organ in which inactivation of a This is the only organ in which inactivation of a single gene, either single gene, either ogg1ogg1 or or myhmyh, is associated , is associated with an age-dependent accumulation of DNA-with an age-dependent accumulation of DNA-8oxoG. 8oxoG. This may reflect a high level of This may reflect a high level of oxidative metabolism or the role of this organ oxidative metabolism or the role of this organ in detoxification.in detoxification.

DNA 8-oxodG accumulates in several mouse DNA 8-oxodG accumulates in several mouse organs when both the MYH and OGG1 organs when both the MYH and OGG1 glycosylases are inactive. glycosylases are inactive. Since Since Xie et al. showed that there is an increased cancer there is an increased cancer incidenceincidence in these organs of in these organs of myhmyh-/--/-ogg1ogg1-/--/- mice, mice, these findings suggest that the accumulation of these findings suggest that the accumulation of oxidized DNA purines play a causative role in oxidized DNA purines play a causative role in cancer development.cancer development.

Xie Y, Yang H, Cunanan C, Okamoto K, Shibata D, Pan J, Barnes DE, Lindahl T, McIlhatton M, Fishel R, Miller JH. Cancer Res. 2004;64:3096.

LiverLiver

Lung Lung Small intestineSmall intestine

Accumulation of 8-oxoG and cancer ?Accumulation of 8-oxoG and cancer ?



2-fold 2-fold (MEFs)(MEFs)

cancercancer

_

_

lymphomaslymphomasGI tractGI tract

Ogg1Ogg1-/--/-

MyhMyh-/--/-

Msh2Msh2-/--/-

•Low ogg1 activity in NSCLC

•High levels 8-oxoG in lung tissues

•OGG1-Cys326 polymorphism

and lung cancer

Hereditary Non Polyposis Colon

Cancerleukemia, lymphoma

mutations in FAP

Ogg1Ogg1-/- -/- MyhMyh-/--/- liver, lungliver, lungSmall intSmall int..

lung,lung,small int.small int.

Ogg1Ogg1-/- -/- MyhMyh-/- -/- Msh2Msh2+/-+/- lunglung ????

kidneyspleenbrain

The absence of a significative accumulation of The absence of a significative accumulation of 8-oxoG in these organs8-oxoG in these organs suggest that there suggest that there might be other DNA repair factors (might be other DNA repair factors (Nucleotide Nucleotide Excision Repair, NEIL1 Excision Repair, NEIL1 and and NEIL2 glycosylasesNEIL2 glycosylases) ) involved in their protection.involved in their protection.

kidneykidney spleenspleen ogg1- myh-

baseline csb-

myhmyh--

ogg1-

ogg1- csb-

Osterod 2002, Oncogene 21: 8232

Fpg s

ensi

tive s

ites

/10

-6 b

p

Osterod 2002, Oncogene 21: 8232

ogg1-

ogg1- csb-

csb-

ogg1- myh-

myhmyh--

liver

Redundancyin the

pathways for

removal of 8-oxoG in the liver

ROS

MTH1MTH1

Oxidized Oxidized dNTPdNTP

8-OH-dGTP8-OH-dGTP2-OH-dATP2-OH-dATP

8-OH-8-OH-dGMPdGMP2-OH-2-OH-dAMPdAMP replicationreplication

errorserrors++

oxidative oxidative DNA DNA

damagedamage

““Mutator Mutator phenotype”phenotype”

Does accumulation

of oxidized DNA bases

contribute to spontaneous

tumorigenesis of MMR-defective

mice?

How much of the association “mutator

phenotype-increased

tumorigenicity” depends on

oxidative DNA-damage?

Construction Construction of a of a

transgenic transgenic mice over-mice over-expressing expressing

hMTH1hMTH1

Cross with an Cross with an msh2msh2-/--/- mice mice

Analysis of Analysis of spontaneous spontaneous

tumorstumors

gWiz+hMTH1 cDNA

CMV promoter + intron

Kan

MscI 245

KpnI 2217

EcoRV

polyA

cDNA of hMTH1cDNA of hMTH1

Number of copies of hMTH1

Transgenic mice

FoundersBamH1

2 5 10 20 40

CMV promoter+intronA

hMTH1 polyAgWiz

GAPDHbra

inbra

in

lung

liver

sple

en

kidney

kidney

Sm

all

int.

ovary

ovary

brain, kidney, ovary, liver, lung,

spleen, small intestine

QuickTime™ e undecompressore TIFF (Non compresso)sono necessari per visualizzare quest'immagine.

hMTH1 is expressed in

several organs of the transgenic

mouse

QuickTime™ e undecompressore TIFF (Non compresso)sono necessari per visualizzare quest'immagine.hMTH1 hMTH1 mousemouse

hMTH1

WT WT mousemouse hMTH1 GAPDH

RT-PCR

Construction Construction of a of a

transgenic transgenic mice over-mice over-expressing expressing

hMTH1hMTH1

hMTH1 is expressed in all the organs

of the transgenic

mouse

tubulin

bra

inbra

in

lung

liver

sple

en

kidney

kidney

Sm

all

int.

ovary

ovary

MTH1

DLD

1D

LD1

MTH1

WT mouse

hMTH1 transgenic mouse

QUESTIONQUESTIONS TO BE S TO BE

ANSWEREANSWEREDD

Will MTH1 overexpression modulate tumorigenicity in msh2-/- mice? Which are the steady-state levels of DNA 8-oxoG in different

organs of msh2-/- mice?

√

√

Can increased cleansing of the Can increased cleansing of the dNTP pool protect from cancer ??dNTP pool protect from cancer ??

Can overexpression of the hMTH1 protein decrease the steady-state levels of DNA 8-

oxoG?

√ Can the overexpression of the hMTH1 protein in the brain provide protection

against oxidative stress induced by neurotoxins (3-NPA for HD; MPTP and

Parkinson’s)

P. FortiniP. FortiniE. ParlantiE. ParlantiE. DogliottiE. Dogliotti

MT. RussoMT. RussoG. De LucaG. De LucaG. AquilinaG. AquilinaM.F. BlasiM.F. BlasiF. ChieraF. Chiera

F. BaroneF. BaroneM. MazzeiM. Mazzei

M. BignamiM. Bignami

ISTITUTO SUPERIORE ISTITUTO SUPERIORE DI SANITA’DI SANITA’

P. KarranP. KarranD. BarnesD. BarnesT. LindahlT. Lindahl

CANCER RESEARCH UK, CANCER RESEARCH UK, SOUTH MIMMSSOUTH MIMMS

Y. NakabeppuY. Nakabeppu M. FuruichiM. FuruichiM. SekiguchiM. Sekiguchi

KYUSHU UNIVERSITY, FUKUOKA

P. DeganP. Degan ISTITUTO NAZIONALE PER LA ISTITUTO NAZIONALE PER LA

RICERCA SUL CANCRO, RICERCA SUL CANCRO, GENOVAGENOVA

H. Te Riele H. Te Riele THE NETHERLANDS CANCER INSTITUTE

AMSTERDAM

J. H. MillerJ. H. MillerUNIVERSITY OF CALIFORNIAUNIVERSITY OF CALIFORNIA

LOS ANGELESLOS ANGELESC. Tiveron L. Tatangelo

TRANSGENIC MICE SERVICE CENTER, ISTITUTO REGINA ELENA,

ROMA