Dommages de l ADN, mutagenèse, carcinogenèse induits par les UV : quel rôle pour les UVA?

Dommages de l ADN, mutagenèse, carcinogenèse induits par les UV : quel rôle pour les UVA? Evelyne SAGE CNRS UMR3348 Institut Curie, Université de Paris-Sud (XI) Orsay (France) Evelyne.Sage@curie.fr 17 e Journées nationales de la Société Française de Photodermatologie Angers, 12 13 mars, 2015

http://www.photobiologie-france.fr Ses missions: coordonner et promouvoir la recherche en Photobiologie sous toutes ses formes promouvoir la formation en Photobiologie promouvoir les activités jointes avec d autres sociétés Ses moyens: organisation de journées scientifiques, thématiques ou non bourses de congrès pour doctorants et post-doctorants bourses de visites pour doctorants et post-doctorants Colloques : Une cinquantaine de membres actifs et membres d honneur avec la Société italienne de photobiologie : 2000, 2004, 2010 avec la Société Française des radicaux Libres, sept. 2008 avec la SFBBM et la Soc Française de Biophysique, nov 2012 Journée de la Photobiologie juin 2013 À venir : Journées Photoprotection, 4-5 juin 2015, Paris Avec le Groupe Français de Photochimie, Photophysique et Photosciences, automne 2015 avec la Société italienne de photobiologie, 2016 Avec la SFPD.

Photobiologie : matière multi- et trans- disciplinaire Nos mots-clés Lésions/réparation de l ADN Photogénotoxicité Photosensibilisation Stress UV Oxygène singulet Photolyase Cryptochromes Photosynthèse UV et microbiologie Protéines photoactives Photobiologie environnementale Spectroscopie abs & fluorescence Chimiluminescence Biphotonique Imagerie de fluorescence Photodiagnostic Thérapie photodynamique Molécules photoactivables Quantum dots Prévention solaire Photoprotection Pigmentation Photovieillissement Antioxydants

Journées Photoprotection 4-5 juin 2015, Paris Programme préliminaire Conférence plénière Jean François Doré : effets biologiques des radiations UV solaires Session populations à risque (enfants, travailleurs ) orateurs: Emmanuel Mahé, Mathieu Boniol Session écrans solaires (fiabilité, aspects réglementaires, écrans et ADN..) orateurs: Laurence Coiffard, Dominique Moyal, Thierry Douki. Session autres types de photoprotection (vêtements, vitd et Photoprotection,..) orateurs: Serge Mordon, François Aubin. Session le futur en photoprotection (mécanismes, et perspectives) orateurs: Marie-Do Galibert.. Communications orales

UV & vitd ESP-ESPD joint symposium

skin cancer UV DNA damage CPDs 6-4 PP Cell Cycle Checkpoints activation * DNA repair * translesion synthesis * signaling oncogenes tumour suppressor genes mutations carcinogenesis

UV mutagenic signature nearly all mutations at bipyrimidines sites a predominance of G.C to A.T transitions some tandem double CC to TT mutations consistent with a role for bipyrimidine photoproducts established by Brash et al. (PNAS 1991) from UVC data and observed in mutated p53 tumor suppressor gene in skin tumors a UVB signature only? CPDs and not 6-4 PP are responsible for the vast majority of mutations induced by UVB in mammalian cells (You et al. JBC 276, 44688, 2001)

mutations in human skin cancers p53 distribution of mutations in Xeroderma pigmentosum (XP) and non-xp skin C T CC TT N XP N XP UVB signature C T and CC TT observable in all 3 types of cancers mutations are localized at specific sites in p53 gene (hot spots) the distribution of hot spots of mutations vary between Basal Cell Carcinoma, Squamous Cell Carcinoma and Malignant Melanoma other genes are frequently and similarly mutated : ex PTCH gene in BCC (Daya-Grosjean et al.) Giglia-Mari & Sarasin, Human Mutation 21, 217, 2003

Terrestrial solar UV radiation Sunlight is a complex mixture of mutagenic wavelengths UV type λ (nm) % UVC 100-280 0 ozone UVB 280-315 5 UVA 315-400 95 0.2 mm 3-4 mm A role for UVA in sunlight-induced mutagenesis and carcinogenesis?

Power (mw/m 2 /nm) 800 Solar spectrum and biological effects 600 400 200 0 UVB UVA Visible 280 300 320 340 360 380 400 420 440 wavelength (nm) direct absorption Photosensitization reactions CPDs 6-4 PP endogenous Photosensitizers ROS ( OH; 1 O 2 ; ROO ) Cellular response to oxidative stress Biomolecules oxidation DNA dntp pool Lipids proteins

UVA radiation induces predominantly Cyclobutane Pyrimidine Dimers and 8oxoG in mammalian cells (even at low doses, i.e. 40-160 kjm-2) UVA SSB oxpyr 8oxoG 3 10 CPDs Damage per 10 6 bases CPDs 8oxoG 1 1 CHO cells Alkaline agarose gels UVA doses (kjm -2 ) Dose reaching the basal layer of the epidermis that corresponds to a solar exposure of 1-2 hrs at zenith in a clear sunny summer day in Paris! Douki, Sage et al Phochem. Photobiol. 1999 - Perdiz, Sage et al J. Biol. Chem. 2000 Douki, Reynaud-Angelin, Cadet & Sage Biochemistry 42, 9221-9226 (2003) Sage, Girard, Reynaud-Angelin, Douki, unpublished data MRC5 V1 & human lymphoblastoids cells HPLC-MS/MS

UVA radiation induces predominantly Cyclobutane Pyrimidine Dimers and 8oxoG in mammalian cells (even at low doses, i.e. 40-160 kjm-2) UVA SSB oxpyr 8oxoG 3 10 CPDs Nevertheless, the yield of CPDs produced by UVA radiation is low : Yields of CPDs: (nb CPDs per Mb per Jm-2) 1 1 UVC=1 UVB: 10-2 UVA: 10-5 CHO cells Alkaline agarose gels 500 kjm -2 UVA needed to produce about the same amount of CPDs as 500 Jm -2 UVB (50 Jcm -2 ) Douki, Sage et al Phochem. Photobiol. 1999 - Perdiz, Sage et al J. Biol. Chem. 2000 Douki, Reynaud-Angelin, Cadet & Sage Biochemistry 42, 9221-9226 (2003) Sage, Girard, Reynaud-Angelin, Douki, unpublished data

UVA-induced CPDs form predominantly at Thymine-Thymine bipyrimidines in the bulk DNA UVB 6-4 TT UVA 6-4 TC <> TT <> TC <> CT Douki, Reynaud-Angelin, Cadet & Sage (2003) Biochemistry 42, 9221-9226 <> CC CHO cells HPLC-MS/MS 0.03 0.02 0.01 0 0 0.01 0.02 lesion/10 6 bases per J.m -2 lesion/10 6 bases per kj.m -2 UVA-photosensitized triplet energy transfer??

Formation of 8oxoG and Thymine-Thymine dimers within human skin biopsies exposed to UVA radiation Induction of CPDs and 6-4 (0-0.2 Jcm -2 ) 6 donors human skin No UVA 8oxoG UVA 8oxoG UVB UVA TT-CPDs UVA Induction of CPDs (0-200 Jcm -2 ) Skin keratinocytes UVA More TT-CPD than 8oxoG induced by UVA in skin of all donors Mouret, Baudouin Cadet, Douki PNAS 103, 13767 (2006)

The sites and frequencies of CPDs formation in cellulo and in vitro are similar TT TT TT APRT exon2, by LM-PCR TT Evidence for CPDs formation by direct absorption of UVA by DNA,. like for UVB or UVC Rochette,. Drouin, Sage. 2003 NAR 31, 2786 Girard, Rochette, Drouin, Sage 2011 J; Phys. Conf. Ser. 261, 012002

Further evidence for a direct photochemical process in UVA-induced CPDs - Observed over the entire UVA range (no UVB artifact!) (Kielbassa et al. (1997) Carcinogenesis, Mouret et al. (2010) Org. Biomol.) - UVA-induced CPDs also in isolated DNA (Quaite et al. 1992; Zhang et al. 1997; Kuluncsics et al. 1999; Jiang et al. 2009; Schuch et al. 2009) - Similar yields in isolated and cellular DNA (Kuluncsics et al. 1999, Perdiz et al. 2000, Mouret et al. (2010) Org. Biomol.; Girard et al JPCS 2011 No involvement of endogenous photosensitizer - DNA, in fact, absorbs UVA (Sutherland & Griffin 1981), as a result of base stacking (Mouret et al. 2010) A 365nm / A 256nm = 10-5 Our yields of CPD in CHO cells: UVC=1 UVA : 10-5 A direct photochemical process - Difference between UVC and UVA photochemistry explained by differences in excited states of bases (Banyasz et al. (2011) J Am Chem Soc)

Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure Sanjay Premi,1 Silvia Wallisch,1 Camila M. Mano,1,2 Adam B. Weiner,1* Antonella Bacchiocchi,3 Kazumasa Wakamatsu,4 Etelvino J. H. Bechara,2,5 Ruth Halaban,3,6 Thierry Douki,7 Douglas E. Brash1,6 Science 20 FEBRUARY 2015 VOL 347 ISSUE 6224 page 842-847 UVA and UVB CPDs continue to increase 4hrs after irradiation ends in melanocytes, not in albino melanocytes dark CPDs account for half of the total CPDs relatively more C-containing CPDs are formed the production of dark CPDs varies with individuals

A role for UVA in solar mutagenesis?

About 15% of CPDs formed by sunlight are produced by UVA CPDs 8oxoG Relative spectral effectiveness in inducing CPDs and 8oxoG for sunlight Calculated from the action spectrum of these photolesions by Epe & col (1997) and from emission spectra of sunlight in Budapest (average) In silico Grof & Sage (from Grof et al JPP-B 2002)

Relatively less mutagenic than UVB Kappes / Rünger et al 2006 JID 126, 667-675 Distribution of mutations induced by UVA (100-250 kjm -2 ) and UVB(150-300 Jm- 2 ) in the hprt gene of primary neonatal human fibroblasts UVA-induced mutations UV mutagenic signature ( CPDs) Ikehata et al. Mut 2003, JID 2008, PPS 2013 : in mouse skin epidermis and dermis (lacz transgene). Large dominance of C to T transitions at Py-mCpG (5 TCG3 ), only 6% GC to TA mutations possibly originating from 8oxoG Agar et al 2004 PNAS 101: UVA fingerprint mutations in human skin SCC and solar keratosis CG to TA mutations at bipyrimidic sites and tandem CC to TT : not only a UVC/UVB signature but also a UVA mutagenic signature! (Sage et al PPS 2012, 11, 74)

UVA exposure, sunbeds use and skin cancer

UVA radiation carcinogenic to humans Ghissassi et al Lancet, 10, 751-752, 2009 In june 2009, scientists at International Agency for Research on Cancer (IARC) classified as «carcinogenic to humans» (Group 1 = like tobacco) : - Solar radiation - Ultraviolet radiation (wavelengths 100-400 nm, encompassing UVA, UVB, UVC) - UV-emitting tanning devices UVB (W/m 2 ) UVA (W/m 2 ) Sunbeds 1.5 3.5 200-350 Tanning lamps 0.2 4 250 550 Sun in july (France) 3.1 56 > 99% UVA The tanning industry sells tanning beds as a safe alternative to UV exposure for both tanning as well as vitamin D biosynthesis.

Epidemiological evidence that UVA radiation is involved in the genesis of cutaneous melanoma: The cases of sunbeds and sunscreen users Ph Autier, JF Doré et al. Current Opinion Oncol 2011, 23, 189-196 Reviews : JF Doré & MC Chignol PPS 2012, 11, 30; Boniol et al BMJ 2012 The use of artificial UV tanning devices (sunbeds) consists mainly of repeated exposure to high UVA doses. Epidemiological studies published over the last years confirmed the association between sunbed use and melanoma : 4 fold increase in melanoma incidence but no increase in mortality from melanoma Sunscreen user for tan acquisition : slightly higher melanoma risk often found, due to extended sun exposure thus leading to similar exposure to UVB and greater exposure to UVA UVA could be involved in the occurrence of nonlife-threatening melanoma. The increasing use of sunbeds and of sunscreens may partly explain why melanoma incidence increases in most light-skinned populations without concomitant increase in mortality.

Mutations in melanoma cells

Melanoma: a genome peppered with mutations A mutational signature of UV exposure Pleasance et al. Nature 2010; 463:1991-196 Sequencing of the genomes of a malignant melanoma and a lymphoblastoid cell line from the same patient (Illumina Genome analyser) 33 345 bases substitutions!!! 24 000 bs were C > T mutations and 92% of C>T occurred at the 3 base of a bipyrimidine site (expected 53% by chance p<0,0001) Over 510 dinucleotide substitutions 360 were CC > TT The 2 nd most frequent mutation type is G > T possibly due to ROS (8oxoG) See also Greenman et al Nature 2007; 446:153158 Laurence et al Nature 2013 doi:10.1038 Nikolaev et al Nature genetics 44, 133, 2012

Melanoma: a genome peppered with mutations A mutational signature of UV exposure Krauthammer et al Nature genetics 44, 1006, 2012 exome sequencing of 147 melanomas and matched germline cells 23 888 missense & 1 596 non-sense mutations, 282 indels Markedly more UV-like C-> T somatic mutations in sun-exposed melanomas than in sun-shielded acral, mucosal and uveal melanomas

Tentative conclusion

In vivo mutation action spectrum follows DNA absorption in the UVA range! Sutherland & Griffin Rad res 1981 Ikehata et al JID 1333, 1850, 2013

Le côté sombre des UVA : Les UVA induisent la formation de dimères cyclobutaniques de pyrimidine (CPDs), essentiellement par absorption par l ADN -> importance en photoprotection Les UVA contribuent pour 15% dans la formation des CPDs induits in vitro par le soleil, mais 200-400 fois plus d UVA que d UVB atteignent la couche basale de l épiderme Combien de CPDs formés à la couche basale de l épiderme sont induits par les UVA? 30%? plus? La mutabilité par CPDs induit est augmentée dans le domaine des UVA comparativement aux UVB (Enninga et al 1986 Carcinogenesis; Robert / Sarasin et al 1995 JID; Kappes / Rünger et al 2006 JID) Probablement due à une activation de p53 et un arrêt de cycle cellulaire réduits après UVA (Rünger et al 2012 PPS) Une empreinte UVA est observée dans les carcinomes SCC et dans les kératoses actiniques solaires (Agar et al 2004 PNAS) La signature mutagène UVA est une signature UV classique

Le côté sombre des UVA : Combien de ces mutations C -> T observées dans les mélanomes ont été induites par les UVA.10, 30% 60%??? Combien d entre elles sont des «driver mutations»??? Combien d entre elles sont impliquées indirectement dans la progression tumorale??? Un rôle sous-estimé des UVA dans les cancers de la peau?

Sage s team Lab. Biology of Radiation CNRS UMR 3348, Institut Curie, Univ. Paris-Sud XI, Orsay (France) on UV Pierre-Marie Girard,senior CNRS research fellow Stefania Francesconi,senior CNRS research fellow Sylvain Martineau, Curie assistant Ludovic Tessier, CNRS technician Dany Graindorge, doc Angela Bellini, doc Daniel Perdiz, doc Zéno Kuluncsics, doc Stas Kozmin, post-doc Anne Reynaud-Angelin Curie IE CEA-Grenoble (France) Thierry Douki Institute of Biophysics, Budapest (Hungary) Pal Grof CHUQ, Université Laval, Québec, Univ of Sherbrooke (Canada) Patrick Rochette, Régen Drouin

Thank you!

Melanin is a photon trap that also acts as an electron-proton photoconductor : Melanin may be carcinogenic as well as protective against cancer Peroxynitrite is also one of the few biologically synthesized molecules capable of exciting an electron to a triplet state. The present experiments show that on a faster time scale, peroxynitrite excites an electron in a melanin fragment to a triplet state that has the high energy of a UV photon.

Sunbed Use Induces the Photoaging-Associated Mitochondrial Common Deletion Jean Krutmann s lab Reimann et al JID 128, 1294-1297, 2008 repetitive UVA irradiationcauses mtdna mutagenesis in vivo and in vitro in human skin cells (Berneburg et al., 2004) and that it can lead to the generation of largescale deletions of mtdna known as the Common Deletion (Pang et al., 1994; Yang et al., 1995; Berneburg et al., 1997; Birch-Machin et al., 1998). More recently, wevidence that the UVA-induced Common Deletion is of pathogenetic relevance for photoaging of human skin (Berneburg et al., 2005). Volunteers without previous sunbed use showed a 2.56-fold increase (95% CI: 0.97 6.78) of the Common Deletion in neck and a 3.58-fold increase (95% CI: 1.44 8.89) of the Common Deletion in buttock skin samples after sunbed use. Participants with previous sunbed use showed no increase.

Melanoma induction by ultraviolet A but not ultraviolet B radiation requires melanin pigment Noonan et al Nature Com 3, 884, 2012 It is showed that melanoma induction by ultraviolet A (320 400 nm) requires the presence of melanin pigment and is associated with oxidative DNA damage within melanocytes. In contrast, ultraviolet B radiation (280 320 nm) initiates melanoma in a pigment-independent manner associated with direct ultraviolet B DNA damage. Same amount of TT CPDS after UVA in both mice skin, pigmented and albino 8oxoG formed in pigmented but not in albino mice

mechanism of UVA-induced CPDs : photosensitization? S 1 64PPs CPDs UVC UVB ISC DNA T 1 Thymine Triplet energy transfer Photosensitizer ISC T 1 UVA S 1 S 0 Lesions /10 4 bases per kj/m 2 0.12 0.08 0.04 Photosensitization of isolated DNA by UVA + acetophenone S 0 CPDs 0 <> TT <> TC <> CT Same damage distribution as induced in cells by UVA no (6-4) photoproduct Douki, Reynaud-Angelin, Cadet & Sage (2003) Biochemistry 42, 9221-9226

CPDs form predominantly at TT sites both in cells and purified DNA upon UVA irradiation and correlate with mutations! CPD formation along exon 2 of APRT (adenine phosphoribosyltransferase) locus in CHO cells was analyzed by Ligation Mediated-PCR UVA UVB T-T 57% 27% T-C 14% 30% C-C 18% 26% C-T 11% 17% Rochette,. Drouin, Sage. 2003 NAR 31, 2786 Girard, Rochette, Drouin, Sage 2011 J; Phys. Conf. Ser. 261, 012002