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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 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.

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

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  1. 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’

  2. 8-oxoguanine abasic site 2-OH-Adenine (5'S)-8,5'-cyclo-2'-deoxyadenosine 8-OH-Adenine Reactive Oxygen Species

  3. 8-oxoG : C anti:anti 8-oxoG : A syn:anti GC ->AT GC ->TA AT ->CG AT ->GC AT ->TA H. Kamiya NAR 31: 517, 2003 2-OH-Ade :T 2-OH-Ade :G GC -> TA AT -> CG Cheng 1992 J.Biol. Chem 267: 166

  4. Pre-replication Replication Post-replication 5’ 5’ 3’ 5’ Base Excision Repair Polymerase selectivity Nucleotide Excision Repair Proofreading by polymerases Translesion synthesis Mismatch Repair MutT hydrolases

  5. Removal of 8-oxoG by Base Excision Repair A ROS Pre-replicative A G* G* G* MYH C C OGG1 G* C Post-replicative + long patch BER

  6. XPC-HR23B interacts with • DNA glycosylases involved • in BER of endogenous lesions (thymine DNA glycosylase, 3-methyl-adenine-DNA- glycosylase) • CSB-/- MEFs are hyper-sensitive 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 • CSB gene product is involved in general genome BER of 8-oxoG • Tuo et al., J.B.C., 2001 276:45772 Nucleotide Excision Repair

  7. G* A* A G/C/A Cleansing of the oxidized dNTP pool by MutT homologues DNA Fujikawa et al., 1999 J.Biol. Chem. 274:18201 Ishibashi T, et al. EMBO Rep. 2003, 4:479 8-oxo-dGTP ROS 8-oxo-dGDP MTH1 NUDT5 8-oxo-dGMP 2-OH-dATP MTH1 2-OH-dAMP

  8. Mismatch formation MutSa: MSH2/MSH6 MutSb: MSH2/MSH3 Mismatch binding Does mismatch repair recognize 8-oxoG-containing mismatches? MutLa: MLH1/PMS2 MutLb: MLH1/PMS1 Mismatch removal Exonuclease Pold/e PCNA RPA RFC DNA resynthesis • 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)

  9. DNA 8-oxoGaccumulates in DNA of MMR-defective cells Steady-state levels hMLH1- msh2-/- 1,6 -/- + cDNA 0,8 +/- 1,2 0,6 +/+ 8-oxodG/ 106 dG 8-oxoG /106 dG 0,8 A2780 0,4 0,4 0,2 H2O2-induced levels 8 10 6 8-oxoG /106 dG 8-oxodG/ 106 dG 6 4 2 2 msh2+/+and msh2-/- MEFs hMLH1- and hMLH1-/cDNA human tumor cells Paolo Degan, IST, Genova

  10. 8-oxoG Incision assay 5’ 8-oxoG 3’ 32P 5’ 3’ C 3’ 5’ extracts from msh2+/+ and msh2-/- MEFs multiple cloning sites “in vitro” BER of 8-oxoG is unaffected by the absence of MMR proteins The accumulation of 8-oxoG in MMR-defective cells cannot be accounted for by a differential efficiency of BER Repair assay in vitro synthesis Extracts from msh2+/+ and msh2-/- MEFs 32P-dNTPs RE digestions and gel electrophoresis

  11. Steady-state 1,8 msh2/ogg1 1,4 1 8-oxodG residues/106 dG msh2-/- ogg1-/- ogg1 msh2 0,6 0,2 +/+ -/- +/+ -/- -/- Msh2 and Ogg1 act independently and their effects are additive msh2-/- ogg1-/- x MEFs Colussi C, et al. Current Biology, 11:912 2002

  12. Pre-replication Replication Post-replication 5’ 5’ 3’ 5’ MTH1 activity 1--6 1--10--50 1 0,8 overexpression of MTH1 0,6 8-oxodG /106 dG 0,4 0,2 0 +/+ -/- Mismatch Repair MutT hydrolases decreased DNA 8-oxoG in MMR-defective cells Colussi C, et al., Current Biology, 11:912 2002 ?? decreased mutagenesis

  13. Mean: 3,1 x 10-6 Mean: 1,8 x 10-7 3° 3 2 1° 1 2° 0 Clone 5 Msh2-/- Clone 2 Clone 5 The mutator phenotype of msh2-/- MEFs is almost abolished by hMTH1 overexpression HPRT gene 60 40 mutation rates x 10-7 20 0 hMTH1 (U/mgprotein) 0,4 3,9 20

  14. AAGGGGGGC 3’ CCCCCC G 5’ 3’ 5’ 3’ AGGGGGG TTCCCCCCG 5’ 12 -1 frameshifts 8 4 0 msh2-/-+hMTH1 hMTH1 overexpression decreases all mutational classes Transversions Frameshifts Transitions 12 mutation rate x 10-7 /cell/generation 8 4 0 msh2-/-

  15. Transitions 2-oxodA C:A* AT>GC 24% 44.1 x Transversions 2-oxodA A:A* AT>TA 12% 61.7 x 8-oxodG A:G* AT>CG 6% 3.2 x 8-oxodG G*:A GC>TA 6% 32 x 2-oxodA G: A* GC>CG 6% 11 x 8-oxodG G*:G 2-OHdATP and 8-oxodGTP can account for a substantial fraction of the mutator phenotype of a MMR-defective cell

  16. Mutator phenotype Microsatellite Instability in vitro 100-fold increase in mutation rates MMR is an important protection against spontaneous mutation and human cancer hMLH1 Hypermethylation SPORADIC Loss of MMR Colorectal Cancer FAMILIAL (HNPCC) Mutations in hMSH2 or hMLH1

  17. BAT26 (A26) DLD1 12 Mutation rate x 10-3 8 DLD1 +MTH1 4 0 SMT15 (G15) DU145 20 Mutation rate x 10-3 10 DU145 +MTH1 0 Overexpression of hMTH1 reduces microsatellite instability of human MMR-defective cell lines ATGCGGTAT AAAAAAAAA(26)CTGATGCA GCATT GGGGGGGGACAC An important component of MSI at mononucleotide A and G runs depends on incorporation of oxidized precursors

  18. 8-OH-dGTP 2-OH-dATP Incorporation of oxidized purines into DNA contributes significantly to the genome instability of MMR- deficient cells ROS MTH1 Oxidized dNTP 8-OH-dGMP 2-OH-dAMP replication errors + oxidative DNA damage 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. “Mutator phenotype”

  19. 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? √ √ √ T C C C C C C G 8- oxoG G G G 3’ 5’ C C C G A G G G G G G C Mouse HPRT -1 frameshifts * AGGGGGC 5’ 3’ 3’ 5’ TCCCCCG C

  20. - - - 0.1 0.1 0.4 0.4 1.2 1.2 3.8 3.8 - - - - - - - - - 0.04 0.04 - - - - - - + + + + + 5’agaacttatag cccccc ttgagcacacagagg3’ aactcgtgtgtctcc5’ 8-oxo dGTP Klenow polymerase 39-mer Incorporationand elongation of 8-oxodGTP opposite C 21-mer 20-mer 19-mer 18-mer 17-mer 16-mer primer 15-mer 8-oxodGTP (mM) dGTP (mM) dNTP(mM) F. Barone

  21. 5’agaacttatag aaaaaa ttgagcacacagagg3’ aactcgtgtgtctcc5’ Klenow polymerase 39-mer Incorporation and elongation of 8-oxodGTP opposite A 16-mer primer 15-mer 8-oxodGTP (nM) - - - - 12 40 120 375 dGTP (mM) - - 375 375 - - - - - - - - - + - + - + - + - + - + dNTP (mM) F. Barone

  22. 5’ cccccc ttgagcacacagagg3’ aactcgtgtgtctcc5’ human polymerase a Incorporation and elongation of 8-oxodGTP opposite C primer 15-mer 8-oxodGTP (mM) - 10 - 3 3 10 30 30 100 100 - - + - + - + - + 10 dGTP (mM) F. Barone G. Maga

  23. 5’ aaaaaa ttgagcacacagagg3’ aactcgtgtgtctcc5’ human polymerase a Incorporation of 8-oxodGTP opposite A No elongation 16-mer 15-mer primer 8-oxodGTP (mM) 10 - - - - 3 3 10 30 30 1 1 - - - - - - - - - - dGTP (mM) 10 10 - 10 - 10 - 10 - 10 - 10 - 10 dTTP (mM) F. Barone G. Maga

  24. AAAAAA Inc. Elong. CCCCCC Inc. Elong. Polymerase family Klenow polymerase polg A + + + + human polymerase a + - + + pold,pole B polh Y DPO4 + - - - Depending on the polymerase 8-oxodGTP be incorporated and elongated in C or A runs

  25. Accumulation of 8-oxoG and cancer ? cancer _ ?? Ogg1-/- 2-fold (liver) biallelic mutations in Familial Adenomatous Polyposis (FAP) _ Myh-/- 4-fold (liver) 2-fold (MEFs) liver, lung, stomach Mth-/- ?? Hereditary Non Polyposis Colon Cancer Leukemia, lymphoma lymphomas GI tract Msh2-/- 2-fold (MEFs)

  26. 2 2 2 brain spleen kidney 1,6 1,6 1,6 1,2 1,2 1,2 8-oxodG residues/106 dG 0,8 0,8 0,8 0,4 0,4 0,4 0 0 0 4 8 12 4 8 12 12 4 8 months months months In wild-type animalssteady-state levels of DNA 8-oxoG do not vary with age WT 2 2 2 small intestine lung liver 1,6 1,6 1,6 1,2 1,2 1,2 8-oxodG residues/106 dG 0,8 0,8 0,8 0,4 0,4 0,4 0 0 0 4 8 12 4 8 12 4 8 12 Osterod et al, Carcinogenesis 2001

  27. Pre-replicative ROS Ogg1-/- G* G* Osterod et al, Carcinogenesis 2001 C OGG1 Post-replicative Ogg1-/- Myh-/- Myh-/- A A 2 2 G* 1,6 1,6 1,2 1,2 8-oxodG residues/106 dG MYH 0,8 0,8 0,4 0,4 0 12 16 4 8 months 4 8 12 16 0 months Levels of 8-oxoG increase in BER-defective mice: liver

  28. Ogg1-/- Myh-/- Myh-/- 2 2 1,6 1,6 1,2 1,2 lung 8-oxodG residues/106 dG 0,8 0,8 0,4 0,4 0 0 4 8 12 16 4 8 12 16 months months 2 2 1,6 1,6 Small intestine 1,2 1,2 8-oxodG residues/106 dG 0,8 0,8 0,4 0,4 0 0 4 8 12 16 4 8 12 16 months months Levels of 8-oxoG increase synergistically in BER-defective 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.

  29. 2 2 2 Myh Ogg1 WT 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 0 0 16 4 8 12 4 8 12 16 4 8 12 2 2 2 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 4 8 12 0 0 4 8 12 16 16 4 8 12 2 2 2 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 0 0 12 4 8 4 8 12 16 16 4 8 12 No accumulation in the levels of DNA 8-oxoG was observed in other organs of Myh-/-Ogg1-/-mice Myh Brain 8-oxodG residues/106 dG Kidney Spleen months

  30. Liver 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. Lung Small intestine 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 incidencein 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.

  31. Accumulation of 8-oxoG and cancer ? cancer • Low ogg1 activity in NSCLC • High levels 8-oxoG in lung tissues • OGG1-Cys326 polymorphism • and lung cancer _ 2-fold (liver) Ogg1-/- liver, lung Small int. lung, small int. Ogg1-/- Myh-/- 4-fold (liver) _ Myh-/- mutations in FAP Ogg1-/- Myh-/- Msh2+/- lung ?? ?? Hereditary Non Polyposis Colon Cancer leukemia, lymphoma 2-fold (MEFs) lymphomas GI tract Msh2-/-

  32. The absence of a significative accumulation of 8-oxoG in these organssuggest that there might be other DNA repair factors (Nucleotide Excision Repair, NEIL1 and NEIL2 glycosylases) involved in their protection. kidney spleen brain ogg1- csb- Fpg sensitive sites /10-6 bp ogg1- csb- baseline ogg1- myh- myh- kidney spleen Osterod 2002, Oncogene 21: 8232

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

  34. 8-OH-dGTP 2-OH-dATP Does accumulation of oxidized DNA bases contribute to spontaneous tumorigenesis of MMR-defective mice? ROS MTH1 Oxidized dNTP 8-OH-dGMP 2-OH-dAMP replication errors + oxidative DNA damage How much of the association “mutator phenotype-increased tumorigenicity” depends on oxidative DNA-damage? “Mutator phenotype”

  35. Construction of a transgenic mice over-expressing 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 Founders EcoRV KpnI 2217 BamH1 cDNA of hMTH1 Analysis of spontaneous tumors

  36. WT mouse hMTH1 GAPDH CMV promoter+intronA hMTH1 polyA gWiz hMTH1 is expressed in several organs of the transgenic mouse RT-PCR Small int. kidney ovary spleen brain lung liver hMTH1 hMTH1 mouse GAPDH brain, kidney,ovary, liver, lung, spleen, small intestine

  37. hMTH1 transgenic mouse Small int. kidney ovary DLD1 spleen brain lung liver MTH1 tubulin MTH1 WT mouse Construction of a transgenic mice over-expressing hMTH1 hMTH1 is expressed in all the organs of the transgenic mouse

  38. √ Can overexpression of the hMTH1 protein decrease the steady-state levels of DNA 8-oxoG? QUESTIONS TO BE ANSWERED √ 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 ??

  39. P. Karran D. Barnes T. Lindahl CANCER RESEARCH UK, SOUTH MIMMS 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 J. H. Miller UNIVERSITY OF CALIFORNIA LOS ANGELES C. Tiveron L. Tatangelo TRANSGENIC MICE SERVICE CENTER, ISTITUTO REGINA ELENA, ROMA Y. Nakabeppu M. Furuichi M. Sekiguchi KYUSHU UNIVERSITY, FUKUOKA H. Te Riele THE NETHERLANDS CANCER INSTITUTE AMSTERDAM

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