Download seminario 11-03

Studio dei meccanismi di
riparazione del danno ossidativo:
modelli “in vitro“ e “in vivo”
11 marzo 2005
Centro Ricerche ENEA Casaccia
M. Bignami
Istituto Superiore di Sanita’
Reactive Oxygen Species
8-oxoguanine
abasic site
NH2
2-OH-Adenine
N
H
HO
N
C
N
N
O
H
8-OH-Adenine
H
H
OH
H
H
(5'S)-8,5'-cyclo-2'-deoxyadenosine
GC -> TA
AT -> CG
Cheng 1992
J.Biol. Chem
267: 166
8-oxoG : C
anti:anti
8-oxoG : A
syn:anti
GC ->AT
GC ->TA
AT ->CG
AT ->GC
AT ->TA
2-OH-Ade :T
2-OH-Ade :G
H. Kamiya NAR
31: 517, 2003
Pre-replication
Replication
Post-replication
5’
5’
3’
5’
Base Excision
Repair
Nucleotide
Excision
Repair
MutT hydrolases
Mismatch Repair
Polymerase selectivity
Proofreading by polymerases
Translesion synthesis
Removal of 8-oxoG by Base Excision Repair
Pre-replicative
ROS
G*
G*
C
OGG1
C
A
A
G*
MYH C
G*
Post-replicative
+ long patch BER
•XPC-HR23B interacts with
DNA glycosylases involved
in BER of endogenous
lesions (thymine DNA
glycosylase, 3-methyladenine-DNA- glycosylase)
•CSB-/- MEFs are hypersensitive to g-irradiation
•CSB -/- exposed to DHEP
show weight reduction
De Waard MCB 2004, 24: 7941
•Primary fibroblasts of CS
patients are defective in repair
of 8-oxoG
Tuo et al., FASEB, 2003 17:668
Nucleotide
Excision
Repair
•CSB gene product is involved
in general genome BER of 8oxoG
Tuo et al., J.B.C., 2001
276:45772
Cleansing of the oxidized dNTP pool by MutT homologues
Fujikawa et al., 1999
DNA
J.Biol. Chem. 274:18201
Ishibashi T, et al.
EMBO Rep. 2003, 4:479
8-oxo-dGTP
G*
RO
S
A
8-oxo-dGDP
MTH1
NUDT5
8-oxo-dGMP
A*
2-OH-dATP
MTH1
2-OH-dAMP
G/C/A
Does mismatch repair recognize 8-oxoG-containing mismatches?
Mismatch
formation
MutSa: MSH2/MSH6
Mismatch
binding
Mismatch
removal
DNA
resynthesis
MutSb: MSH2/MSH3
MutLa: MLH1/PMS2
MutLb: MLH1/PMS1
Exonuclease
Pold/e
PCNA
RPA
RFC
• In the absence of MSH2/MSH6 mutation rates are highly elevated. When cells are grown
anaerobically, the rates are decreased (PNAS 1998, 95:15487)
• MSH2 and MSH6 are required for removal of adenine misincorporated opposite 8-oxoG
(Mol Cell. 1999;4:439)
DNA 8-oxoG accumulates in DNA of MMR-defective cells
Steady-state levels
8-oxoG /106 dG
and
hMLH1-/cDNA
human tumor cells
+/0,6
0,4
0,2
+/+
1,6
8-oxodG/ 106 dG
-/-
0,8
hMLH1-
hMLH1-
msh2-/-
+ cDNA
and
msh2-/- MEFs
1,2
0,8
A2780
msh2+/+
0,4
H2O2-induced levels
8-oxoG /106 dG
6
2
Paolo Degan, IST, Genova
8-oxodG/ 106 dG
8
10
6
4
2
Incision assay
The
accumulation
of 8-oxoG in
MMRdefective
cells cannot
be accounted
for by a
differential
efficiency of
BER
32P5’
3’
8-oxoG
C
“in vitro” BER of 8-oxoG is
unaffected by the absence
of MMR proteins
3’
5’
extracts from msh2+/+
and msh2-/- MEFs
Repair assay
8-oxoG
5’
3
’
in vitro
synthesis
Extracts from msh2+/+
and msh2-/- MEFs
32P-dNTPs
multiple
cloning sites
RE digestions and
gel electrophoresis
Msh2 and Ogg1 act independently and their
effects are additive
Steady-state
ogg1-/-
1,8
8-oxodG residues/106 dG
msh2-/-
x
msh2-/- ogg1-/-
1,4
msh2/ogg
1
1
0,6
msh2 ogg1
0,2
+/+ -/- +/+ -/-
-/-
MEFs
Colussi C, et al. Current Biology, 11:912 2002
Pre-replication
Replication
Post-replication
5’
5’
Mismatch Repair
3’
5’
MutT hydrolases
overexpression
of MTH1
decreased
DNA 8-oxoG
in MMRdefective cells
Colussi C, et al., Current Biology, 11:912 2002
8-oxodG /106 dG
MTH1
activity
1--6
1--10--50
+/+
-/-
1
0,8
0,6
0,4
0,2
0
?? decreased mutagenesis
The mutator phenotype of msh2-/- MEFs is almost
abolished by hMTH1 overexpression
HPRT gene
Mean: 3,1 x 10-6
Mean: 1,8 x 10-7
60
3
mutation rates x 10-7
3°
2
40
1
1°
20
0
2°
Clone 5
0
Msh2-/hMTH1
(U/mgprotein)
0,4
Clone 2 Clone 5
3,9
20
hMTH1 overexpression decreases all
mutational classes
3’
AAGGGGGGC 3’
5’
CCCCCC G
mutation rate x 10-7
/cell/generation
5’
3’
12
12
8
8
4
4
0
0
msh2-/-
AGGGGGG
TTCCCCCCG 5’
-1 frameshifts
msh2-/- +hMTH1
Transitions
24%
44.1 x
2-oxodA
C:A*
AT>TA
12%
61.7 x
2-oxodA
A:A*
AT>CG
6%
3.2 x
8-oxodG
A:G*
GC>TA
6%
32 x
8-oxodG
2-oxodA
G*:A
G: A*
GC>CG
6%
11 x
8-oxodG
G*:G
AT>GC
Transversions
2-OHdATP and 8-oxodGTP can account for a substantial
fraction of the mutator phenotype of a MMR-defective cell
MMR is an important protection against
spontaneous mutation and human cancer
Mutator phenotype
hMLH1
Hypermethylation
Microsatellite
Instability
in vitro
100-fold increase
in mutation rates
Loss of
MMR
SPORADI
C
FAMILIAL
(HNPCC)
Colorectal
Cancer
Mutations in
hMSH2 or hMLH1
Mutation rate x 10-3
Overexpression of hMTH1 reduces microsatellite instability
of human MMR-defective cell lines
1
2
8
DLD1
BAT26 (A26)
ATGCGGTAT AAAAAAAAA(26) CTGATGCA
DLD1
+
4
MTH1
Mutation rate x 10-3
0
DU145
SMT15 (G15)
20
10
GCATT GGGGGGGG ACAC
DU145
+MTH1
0
An important component of MSI at mononucleotide A and G
runs depends on incorporation of oxidized precursors
Incorporation
of oxidized
purines into
DNA
contributes
significantly to
the genome
instability of
MMR- deficient
cells
8-OH-dGTP
2-OH-dATP
RO
S
MTH1
8-OH-dGMP
2-OH-dAMP
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.
Oxidized
dNTP
replication
errors
+
oxidative
DNA damage
“Mutator phenotype”
TCCCCCCG
8GGG
oxoG
3’
5’
CCCG
AGGGGGGC
√ How easy is it to incorporate and
Mouse HPRT
elongate an 8-oxodGTP?
-1 frameshifts
5’
*
AGGGGGC
3’
3’
T CCCCCG
C
5’
√ Where do the frameshift occur?
√
Frameshifts in runs of A and Gfrom which oxidized triphosphates?
agaacttatag cccccc ttgagcacacagagg3’
aactcgtgtgtctcc5’
5’
8-oxo
dGTP
Klenow
polymerase
39-mer
Incorporation
and
elongation of
8-oxodGTP
opposite C
21-mer
20-mer
19-mer
18-mer
17-mer
16-mer
15-mer
primer
8-oxodGTP (mM) dGTP (mM)
dNTP(mM)
0.1 0.1 0.4
- - -
+
-
0.4
+
1.2
1.2
3.8 3.8
-
-
-
+
-
-
0.04
+
-
0.04
+
F. Barone
agaacttatag aaaaaa ttgagcacacagagg3’
aactcgtgtgtctcc5’
5’
Klenow
polymerase
39-mer
Incorporation
and
elongation of
8-oxodGTP
opposite A
16-mer
15-mer
primer
8-oxodGTP (nM) dGTP (mM) -
-
375 375 -
-
-
-
-
-
-
-
-
+
-
+
-
+
-
+
-
+
dNTP (mM)
-
-
+
12
-
40
120
375
F. Barone
cccccc ttgagcacacagagg3’
aactcgtgtgtctcc5’
5’
human
polymerase a
Incorporation
and
elongation of
8-oxodGTP
opposite C
primer
15-mer
8-oxodGTP (mM)
-
3
3
10
dGTP (mM)
-
-
+
-
10
30
30
+
-
+
100 100
-
+
10
F. Barone
G. Maga
5’
aaaaaa ttgagcacacagagg3’
aactcgtgtgtctcc5’
human
polymerase a
Incorporation
of 8oxodGTP
opposite A
No
elongation
16-mer
15-mer
primer
8-oxodGTP (mM) -
-
-
-
1
1
3
3
10
dGTP (mM)
-
-
10
10
-
-
-
-
dTTP (mM)
-
10
-
-
10
-
10
10
10
30
30
-
-
-
-
-
10
-
10
F. Barone
G. Maga
CCCCCC
Polymerase
family
AAAAAA
Inc.
Elong.
Inc.
Elong.
A
Klenow
polymerase
+
+
+
+
pold,pole
B
human
polymerase a
+
+
+
-
polh
Y
DPO4
-
-
+
-
polg
Depending on the polymerase
8-oxodGTP be incorporated and elongated in C or A runs
Accumulation of 8-oxoG and cancer ?
cancer
Ogg1-/-
2-fold (liver)
Myh-/-
4-fold (liver)
Mth-/-
2-fold (MEFs)
Msh2-/-
2-fold (MEFs)
_
_
liver, lung,
stomach
lymphomas
GI tract
??
biallelic mutations in
Familial Adenomatous
Polyposis (FAP)
??
Hereditary Non
Polyposis Colon
Cancer
Leukemia, lymphoma
In wild-type animals steady-state levels
of DNA 8-oxoG do not vary with age
8-oxodG residues/106 dG
WT
2
small intestine
1,6
1,6
2
lung
1,2
1,2
0,8
0,8
0,8
0,4
0,4
0,4
4
8
12
4
8
12
2
2
1,6
0
brain
1,6
liver
1,6
1,2
0
8-oxodG residues/106 dG
2
0
4
spleen
1,6
1,2
1,2
0,8
0,8
0,8
0,4
0,4
0,4
4
8
months
12
0
12
2
1,2
0
8
4
8
months
12
0
kidney
4
8
12
months
Osterod et al, Carcinogenesis 2001
Levels of 8-oxoG increase in BER-defective
mice: liver
Pre-replicative
Ogg1-/-
ROS
G*
G*
C
Osterod et al,
Carcinogenesis 2001
OGG1
A
A
G*
MYH
8-oxodG residues/106 dG
Post-replicative
Ogg1-/-
Myh-/-
2
1,6
1,6
1,2
1,2
0,8
0,8
0,4
0
Myh-/-
2
0,4
4
8
months
12
16
0
4
8
12
months
16
Myh-/-
8-oxodG residues/106 dG
2
Ogg1-/Myh-/-
2
1,6
1,6
1,2
1,2
0,8
0,8
0,4
0,4
lung
0
4
8
12
0
16
4
months
8-oxodG residues/106 dG
Levels of 8oxoG increase
synergistically
in BERdefective mice
8
12
16
months
2
2
1,6
1,6
Small 1,2
intestine
0,8
1,2
0,8
0,4
0,4
0
0
4
8
12
months
16
4
8
12
16
months
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.
No accumulation in the levels of DNA 8-oxoG was
observed in other organs of Myh-/-Ogg1-/- mice
2
WT
1,6
8-oxodG residues/106 dG
2
2
Myh
1,6
1,2
1,2
1,2
0,8
0,8
0,8
0,4
0,4
0,4
0
2
4
8
12
20
4
8
12
16
20
1,6
1,6
1,6
1,2
1,2
1,2
0,8
0,8
0,8
0,4
0,4
0,4
0
2
4
8
12
1,6
0
2
4
8
12
16
0
2
1,6
1,6
1,2
1,2
0,8
0,8
0,8
0,4
0,4
0,4
1,2
0
4
8
12
0
4
8
months
Myh
Ogg1
1,6
12
16
0
Brain
4
8
12
16
Kidney
4
8
12
16
Spleen
4
8
12
16
Liver This is the only organ in which inactivation of a single
gene, either ogg1 or myh, is associated with an agedependent accumulation of DNA-8oxoG. This may
reflect a high level of oxidative metabolism or the role
of this organ in detoxification.
Lung
DNA 8-oxodG accumulates in several mouse organs
Small intestine when both the MYH and OGG1 glycosylases are
inactive. Since Xie et al. showed that there is an
increased cancer incidence in these organs of myh-/ogg1-/- mice, these findings suggest that the
accumulation of oxidized DNA purines play a causative
role in 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.
Accumulation of 8-oxoG and cancer ?
Ogg1-/-
Ogg1-/- Myh-/Myh-/-
liver, lung
Small int.
4-fold
(liver)
Ogg1-/- Myh-/- Msh2+/Msh2-/-
_
2-fold
(liver)
??
2-fold
(MEFs)
lung,
small int.
cancer
•Low ogg1 activity in NSCLC
•High levels 8-oxoG in lung
tissues
•OGG1-Cys326 polymorphism
and lung cancer
_
mutations in FAP
lung
??
lymphomas
GI tract
Hereditary Non Polyposis
Colon Cancer
leukemia, lymphoma
The absence of a significative accumulation of 8-oxoG
in these organs suggest that there might be other DNA
repair factors (Nucleotide Excision Repair, NEIL1 and
NEIL2 glycosylases) involved in their protection.
Fpg sensitive sites /10-6 bp
kidney
spleen
brain
ogg1- csb-
baseline
kidney
spleen
ogg1-
csb-
ogg1- myh- myhOsterod 2002, Oncogene 21: 8232
ogg1csb-
Redundancy
in the
pathways
for removal
of 8-oxoG in
the liver
ogg1myh-
ogg1-
myh-
csb-
liver
Osterod 2002, Oncogene 21: 8232
Does
accumulation of
oxidized DNA
bases
contribute to
spontaneous
tumorigenesis
of MMRdefective mice?
8-OH-dGTP
2-OH-dATP
RO
S
MTH1
8-OH-dGMP
2-OH-dAMP
How much of the
association “mutator
phenotype-increased
tumorigenicity”
depends on oxidative
DNA-damage?
Oxidized
dNTP
replication
errors
+
oxidative
DNA damage
“Mutator phenotype”
Construction
of a transgenic
mice overexpressing
hMTH1
Number of copies
of hMTH1
MscI 245
Transgenic mice
CMV
promoter +
intron
Kan
gWiz+
hMTH1 cDNA
Cross with an
msh2-/- mice
2 5 10 20 40
polyA
EcoRV
KpnI BamH1
2217
cDNA of hMTH1
Analysis of
spontaneous
tumors
Founders
CMV promoter+intronA
hMTH1
polyA
ovary
Small int.
kidney
spleen
liver
lung
RT-PCR
brain
hMTH1 is
expressed in
several organs
of the
transgenic
mouse
gWiz
hMTH1
hMTH1
mouse
brain, kidney, ovary,
liver, lung, spleen,
small intestine
GAPDH
QuickTime™ e un decompressore TIFF (Non compresso) sono necessari per visualizzare quest'immagine.
WT
mouse
hMTH1
GAPDH
DLD1
brain
lung
spleen
liver
kidney
Small int.
hMTH1 transgenic mouse
ovary
Construction
of a transgenic
mice overexpressing
hMTH1
MTH1
tubulin
hMTH1 is
expressed in
all the organs
of the
transgenic
mouse
MTH1
WT mouse
QUESTION
S TO BE
ANSWERE
D
√
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)
√
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 dNTP
pool protect from cancer ??
P. Fortini
E. Parlanti
E. Dogliotti
MT. Russo
G. De Luca
G. Aquilina
M.F. Blasi
F. Chiera
F. Barone
M. Mazzei
M. Bignami
ISTITUTO SUPERIORE
DI SANITA’
P. Degan
ISTITUTO NAZIONALE PER LA
RICERCA SUL CANCRO,
GENOVA
P. Karran
D. Barnes
T. Lindahl
CANCER RESEARCH UK,
SOUTH MIMMS
J. H. Miller
C. Tiveron
L. Tatangelo
TRANSGENIC MICE SERVICE
CENTER, ISTITUTO REGINA ELENA,
ROMA
UNIVERSITY OF CALIFORNIA
LOS ANGELES
Y. Nakabeppu
M. Furuichi
M. Sekiguchi
KYUSHU UNIVERSITY,
FUKUOKA
H. Te Riele
THE NETHERLANDS
CANCER INSTITUTE
AMSTERDAM