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)

17

Journées nationales de la Société Française de Photodermatologie Angers, 12 – 13 mars, 2015

Evelyne.Sage@curie.fr

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 :

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 ….

Une cinquantaine de membres actifs et membres d’honneur

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

Photobiologie : matière multi- et trans- disciplinaire

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

Cell Cycle Checkpoints

activation

*

DNA repair

*

translesion synthesis

*

signaling

DNA

damage mutations carcinogenesis

skin cancer

UV

oncogenes

tumour suppressor genes

CPDs 6-4 PP

UV mutagenic signature UV mutagenic signature

consistent with a role for bipyrimidine photoproducts

• nearly all mutations at bipyrimidines sites

• a predominance of G.C to A.T transitions

• some tandem double CC to TT mutations

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

Giglia-Mari & Sarasin, Human Mutation 21, 217, 2003

p53 distribution of mutations in Xeroderma pigmentosum (XP) and non-XP skin

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

C → T CC → TT

N XP N XP

UV type λλλλ (nm) % UVC 100-280 0 UVB 280-315 5 UVA 315-400 95

Terrestrial solar UV radiation

ozone

0.2 mm 3-4 mm

A role for UVA in sunlight-induced mutagenesis and carcinogenesis?

Sunlight is a complex mixture of mutagenic wavelengths

UVB UVA Visible

280 300 320 340 360 380 400 420 440

0 200 400 600 800

wavelength (nm)

Po w er ( m W /m

/n m ) Solar spectrum and biological effects

(OH;

ROS

endogenous Photosensitizers

direct absorption Photosensitization reactions

Biomolecules oxidation

DNA dNTP pool

Lipids proteins

CPDs 6-4 PP

UVA radiation induces predominantly Cyclobutane Pyrimidine Dimers and 8oxoG in mammalian cells

(even at low doses, i.e. 40-160 kJm-2)

1 1

3

10

SSB oxPyr 8oxoG CPDs

UVA

CHO cells Alkaline agarose gels

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 !

MRC5 V1 & human lymphoblastoids cells

HPLC-MS/MS UVA doses (kJm^-2)

Damage per 10⁶ bases

CPDs

8oxoG

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

1 1

3

10

SSB oxPyr 8oxoG CPDs

UVA

CHO cells Alkaline agarose gels

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

Nevertheless, the yield of CPDs produced by UVA radiation is low :

Yields of CPDs:

(nb CPDs per Mb per Jm-2)

UVC=1 UVB: 10

UVA: 10

500 kJm^-2UVA needed to produce about the same amount of CPDs as 500 Jm^-2UVB (50 Jcm^-2)

UVA radiation induces predominantly Cyclobutane Pyrimidine Dimers and 8oxoG in mammalian cells

(even at low doses, i.e. 40-160 kJm-2)

Douki, Reynaud-Angelin, Cadet

& Sage (2003) Biochemistry 42, 9221-9226

6-4 TT

0 0.01

0.02 0.03

lesion/10⁶ bases per J.m^-2

0 0.01 0.02

<> CC

<> CT

<> TC

<> TT 6-4 TC

lesion/10⁶ bases per kJ.m^-2

UVB UVA

UVA-photosensitized triplet energy transfer ??

UVA-induced CPDs form predominantly at Thymine-Thymine bipyrimidines in the bulk DNA

CHO cells HPLC-MS/MS

Formation of 8oxoG and Thymine-Thymine dimers within human skin biopsies exposed to UVA radiation

Mouret, Baudouin …Cadet, Douki PNAS 103, 13767 (2006) Induction of CPDs and 6-4 (0-0.2 Jcm^-2)

Induction of CPDs (0-200 Jcm^-2)

6 donors human skin

More TT-CPD than 8oxoG induced by UVA in skin of all donors UVB

UVA

UVA

No UVA 8oxoG UVA 8oxoG UVA TT-CPDs

Skin

keratinocytes

The sites and frequencies of CPDs formation in cellulo and in vitro are similar

APRT exon2, by LM-PCR TT

TT

TT

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

- 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 - Observed over the entire UVA range (no UVB artifact!)

(Kielbassa et al. (1997) Carcinogenesis, Mouret et al. (2010) Org. Biomol.)

No involvement of endogenous photosensitizer

- Difference between UVC and UVA photochemistry explained by differences in excited states of bases (Banyasz et al. (2011) J Am Chem Soc)

A direct photochemical process - 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

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

• 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

UVA and UVB

A role for UVA in solar mutagenesis?

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)

CPDs 8oxoG

Grof & Sage (from Grof et al JPP-B 2002)

About 15% of CPDs formed by sunlight are produced by UVA

In silico

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

UV mutagenic signature ( CPDs)

UVA-induced mutations

Relatively less mutagenic than UVB

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)

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

UVA exposure, sunbeds use and skin cancer

UVA radiation carcinogenic to humans

The tanning industry ‘‘sells’’ tanning beds as a safe alternative to UV exposure for both tanning as well as vitamin D biosynthesis.

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

Ghissassi et al Lancet, 10, 751-752, 2009

UVB (W/m²) UVA (W/m²)

Sunbeds 1.5 – 3.5 200 - 350

Tanning lamps 0.2 – 4 250 – 550

Sun in july (France) 3.1 56

> 99% UVA

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

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.

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

Mutations in melanoma cells

Melanoma: a genome peppered with mutations A mutational signature of UV exposure

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 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

most frequent mutation type is G > T possibly due to ROS

(8oxoG)

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.

Reimann et al JID 128, 1294-1297, 2008

Sunbed Use Induces the Photoaging-Associated Mitochondrial Common Deletion

Jean Krutmann’s lab

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 large- scale 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

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

Noonan et al Nature Com 3, 884, 2012

Photosensitizer

Triplet energy transfer

S

S

UVA

ISC T

Photosensitization of isolated DNA by UVA + acetophenone

0 0.04 0.08 0.12

<> TT <> TC <> CT

Lesions /104 bases per kJ/m2

Same damage distribution as induced in cells by UVA no (6-4) photoproduct …

mechanism of UVA-induced CPDs : photosensitization?

Douki, Reynaud-Angelin, Cadet & Sage (2003) Biochemistry42, 9221-9226

DNA

ISC T

S

UVC UVB

S

64PPs CPDs

CPDs Thymine

Rochette, …. Drouin,… Sage. 2003 NAR 31, 2786

Girard, …Rochette, Drouin, Sage 2011 J; Phys. Conf. Ser. 261, 012002

CPD formation along exon 2 of APRT (adenine phosphoribosyltransferase) locus in CHO cells was analyzed by Ligation Mediated-PCR

CPDs form predominantly at TT sites both in cells and purified DNA upon UVA irradiation

UVA UVB T-T 57% 27%

T-C 14% 30%

C-C 18% 26%

C-T 11% 17%

… and correlate with mutations !