Download - 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)
Klenow Klenow polymerasepolymerase
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
human human polymerase polymerase
8-oxodGTP (M)
primer 15-mer16-mer
F. BaroneF. BaroneG. MagaG. Maga
human human polymerase polymerase
Klenow Klenow polymerasepolymerase
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)
4-fold 4-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 (liver)(liver)
4-fold 4-fold (liver)(liver)
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