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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