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

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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’. 8-oxoguanine. abasic site. 2-OH-Adenine. (5 'S)- 8,5 '-cyclo -2 ' -deoxyadenosine. 8-OH-Adenine. - PowerPoint PPT Presentation

Text of Studio dei meccanismi di riparazione del danno ossidativo: modelli “in vitro“ e “in vivo”

  • Studio dei meccanismi di riparazione del danno ossidativo: modelli in vitro e in vivo

    11 marzo 2005Centro Ricerche ENEA CasacciaM. Bignami Istituto Superiore di Sanita

  • Reactive Oxygen Species

  • GC -> TAAT -> CGCheng 1992 J.Biol. Chem267: 166

  • MutT hydrolasesMismatch Repair

  • Post-replicativeG*MYHAAG*COGG1G*CG*ROSPre-replicativeCRemoval of 8-oxoG by Base Excision Repair+ long patch BER

  • NucleotideExcisionRepairCSB-/- MEFs are hyper-sensitive to g-irradiationCSB -/- exposed to DHEP show 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 276:45772XPC-HR23B interacts with DNA glycosylases involved in BER of endogenous lesions (thymine DNA glycosylase, 3-methyl-adenine-DNA- glycosylase)

  • Cleansing of the oxidized dNTP pool by MutT homologuesFujikawa et al., 1999 J.Biol. Chem. 274:18201 ROS8-oxo-dGTP8-oxo-dGDP8-oxo-dGMPDNAMTH1NUDT5Ishibashi T, et al. EMBO Rep. 2003, 4:4792-OH-dATP2-OH-dAMPMTH1

  • Mismatch removalDNA resynthesisMutLa: MLH1/PMS2MutLb: MLH1/PMS1Pold/ePCNARPARFCExonucleaseDoes mismatch repair recognize 8-oxoG-containing mismatches? MSH2 and MSH6 are required for removal of adenine misincorporated opposite 8-oxoG (Mol Cell. 1999;4:439) In the absence of MSH2/MSH6 mutation rates are highly elevated. When cells are grown anaerobically, the rates are decreased (PNAS 1998, 95:15487)

  • hMLH1- and hMLH1-/cDNA human tumor cellsmsh2+/+ and msh2-/- MEFsPaolo Degan, IST, Genova

  • in vitro BER of 8-oxoG is unaffected by the absence of MMR proteinsExtracts from msh2+/+ and msh2-/- MEFs32P-dNTPs in vitro synthesisRE digestions and gel electrophoresisRepair assayThe accumulation of 8-oxoG in MMR-defective cells cannot be accounted for by a differential efficiency of BER

  • Msh2 and Ogg1 act independently and their effects are additiveColussi C, et al. Current Biology, 11:912 2002msh2-/-ogg1-/-xMEFs

  • decreased DNA 8-oxoGin MMR-defective cells ?? decreased mutagenesis Colussi C, et al., Current Biology, 11:912 2002MutT hydrolasesMismatch Repair

  • The mutator phenotype of msh2-/- MEFs is almost abolished by hMTH1 overexpressionhMTH1 (U/mgprotein) 0204060mutation rates x 10-70,43,920HPRT gene

  • hMTH1 overexpression decreases all mutational classesmutation rate x 10-7 /cell/generationTransversionsFrameshiftsTransitionsmsh2-/-04812

  • AT>GC24%44.1 xAT>TA 12% 61.7 xTransitionsTransversionsAT>CG 6% 3.2 xC:A*2-oxodAGC>TA 6% 32 xGC>CG 6% 11 x2-OHdATP and 8-oxodGTP can account for a substantial fraction of the mutator phenotype of a MMR-defective cell2-oxodAA:A*A:G*8-oxodG8-oxodGG*:AG: A*2-oxodA8-oxodGG*:G

  • MMR is an important protection against spontaneous mutation and human cancerhMLH1 HypermethylationMutations in hMSH2 or hMLH1SPORADICFAMILIAL (HNPCC)Colorectal CancerLoss of MMR

  • Overexpression of hMTH1 reduces microsatellite instability of human MMR-defective cell linesAn important component of MSI at mononucleotide A and G runs depends on incorporation of oxidized precursorsGCATT GGGGGGGG ACACATGCGGTAT AAAAAAAAA(26) CTGATGCA

  • ROSMTH1Oxidized dNTP Incorporation of oxidized purines into DNA contributes significantly to the genome instability of MMR- deficient cellsRusso 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-dGTP2-OH-dATP8-OH-dGMP2-OH-dAMPreplicationerrors+oxidative DNA damageMutator phenotype

  • A G G G G G G CC C C GG G GT C C C C C C G8-oxoG53Mouse HPRT-1 frameshifts53T CCCCCG35AGGGGGC*C

  • Incorporationand elongation of 8-oxodGTPopposite C aactcgtgtgtctcc55agaacttatag cccccc ttgagcacacagagg3primer16-mer15-mer17-mer18-mer19-mer20-mer21-merdGTP (mM)dNTP(mM)8-oxodGTP (mM) Klenow polymerase39-mer8-oxodGTPF. Barone

  • Incorporation and elongation of 8-oxodGTPopposite A aactcgtgtgtctcc55agaacttatag aaaaaa ttgagcacacagagg3dGTP (mM)dNTP (mM)--12--+-+--375-375---+--40-+120---+375---+8-oxodGTP (nM) Klenow polymeraseprimer16-mer15-mer39-merF. Barone

  • aactcgtgtgtctcc55 cccccc ttgagcacacagagg3dGTP (mM)+--+-+10-+--3 1030-31030100100Incorporationand elongation of 8-oxodGTP opposite Chuman polymerase a8-oxodGTP (mM) primer15-merF. BaroneG. Maga

  • dGTP (mM)----10----3 1030-3103010---11dTTP (mM)-------- 10 10 10 10 10 10 aactcgtgtgtctcc55 aaaaaa ttgagcacacagagg3Incorporation of 8-oxodGTP opposite A

    No elongationhuman polymerase a8-oxodGTP (mM) primer15-mer16-merF. BaroneG. Maga

  • human polymerase aKlenow polymerasePolymerase familyABDPO4YCCCCCCInc.Elong.++-++-++++--AAAAAAInc.Elong.polgpolhpold,poleDepending on the polymerase 8-oxodGTP be incorporated and elongated in C or A runs

  • 2-fold (liver)4-fold (liver)2-fold (MEFs)2-fold (MEFs)cancer__liver, lung, stomachlymphomasGI tractOgg1-/-Myh-/-Mth-/-Msh2-/-????Hereditary Non Polyposis Colon CancerLeukemia, lymphomabiallelic mutations in Familial Adenomatous Polyposis (FAP)Accumulation of 8-oxoG and cancer ?

  • WTliver00,40,81,21,62481200,40,81,21,6248128-oxodG residues/106 dG0,40,81,21,6204812small intestinelungIn wild-type animals steady-state levels of DNA 8-oxoG do not vary with ageOsterod et al, Carcinogenesis 2001

  • Levels of 8-oxoG increase in BER-defective mice: liver

  • Levels of 8-oxoG increase synergistically in BER-defective miceRusso 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.

  • BrainKidneySpleenmonths8-oxodG residues/106 dGMyhNo accumulation in the levels of DNA 8-oxoG was observed in other organs of Myh-/-Ogg1-/- mice

  • This is the only organ in which inactivation of a single gene, either ogg1 or myh, is associated with an age-dependent accumulation of DNA-8oxoG. This may reflect a high level of oxidative metabolism or the role of this organ in detoxification. DNA 8-oxodG accumulates in several mouse organs 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.LiverLung Small intestine

  • Accumulation of 8-oxoG and cancer ?2-fold (liver)4-fold (liver)2-fold (MEFs)cancer__lymphomasGI tractOgg1-/-Myh-/-Msh2-/-Low ogg1 activity in NSCLCHigh levels 8-oxoG in lung tissuesOGG1-Cys326 polymorphismand lung cancerHereditary Non Polyposis Colon Cancerleukemia, lymphomamutations in FAPOgg1-/- Myh-/-liver, lungSmall int.lung,small int.Ogg1-/- Myh-/- Msh2+/-lung????

  • kidneyspleenbrainThe 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.kidneyspleenogg1- myh-baselinecsb-myh-ogg1- ogg1- csb-Osterod 2002, Oncogene 21: 8232Fpg sensitive sites /10-6 bp

  • Osterod 2002, Oncogene 21: 8232ogg1-ogg1- csb-csb-ogg1- myh-myh-liverRedundancyin the pathways for removal of 8-oxoG in the liver

  • ROSMTH1Oxidized dNTP8-OH-dGTP2-OH-dATP8-OH-dGMP2-OH-dAMPreplicationerrors+oxidative DNA damageMutator phenotypeDoes 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 of a transgenic mice over-expressing hMTH1Cross with an msh2-/- miceAnalysis of spontaneous tumors gWiz+hMTH1 cDNA CMV promoter + intron KanMscI 245 KpnI 2217 EcoRV polyA cDNA of hMTH1Number of copies of hMTH1 Transgenic miceFoundersBamH12 5 10 20 40

  • CMV promoter+intronAhMTH1polyAgWizGAPDHbrainlungliverspleenkidneySmall int.ovarybrain, kidney, ovary, liver, lung, spleen, small intestine hMTH1 is expressed in several organs of the transgenic mousehMTH1 mousehMTH1RT-PCR

  • Construction of a transgenic mice over-expressing hMTH1hMTH1 is expressed in all the organs of the transgenic mouse

  • QUESTIONS TO BE ANSWEREDWill 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 ??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 Parkinsons)

  • P. FortiniE. ParlantiE. Dogliotti

    MT. RussoG. De LucaG. AquilinaM.F. BlasiF. Chiera

    F. BaroneM. MazzeiM. Bignami

    ISTITUTO SUPERIORE DI SANITA P. KarranD. BarnesT. LindahlCANCER RESEARCH UK, SOUTH MIMMSY. Nakabeppu M. FuruichiM. Sekiguchi KYUSHU UNIVERSITY, FUKUOKAP. Degan ISTITUTO NAZIONALE PER LA RICERCA SUL CANCRO, GENOVAH. Te Riele THE NETHERLANDS CANCER INSTITUTEAMSTERDAMJ. H. MillerUNIVERSITY OF CALIFORNIALOS ANGELESC. Tiveron L. Tatangelo TRANSGENIC MICE SERVICE CENTER, ISTITUTO REGINA ELENA, ROMA