Poly(ADP-ribose) in the skin and in malignant melanomas

PAR in he skin and in melanomas His ol. His opa hol. 1 1 Poly(ADP- ibose) in he skin and in melanomas Zsuzsanna Géhl1,2*, Pé e Bai1,3*, Edina Bodná 4, Gab iella Em i4, É a Remenyik4, János Néme h2, Pál Ge gely1,3, László Vi ág1,3,# and É a Szabó4 1Depa men o Medical Chemis y, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Deb ecen, Hunga y 2Semmelweis Uni e si y, Depa men o Oph halmology, Budapes , Hunga y 3Cell Biology and Signaling Resea ch G oup o he Hunga ian Academy o Sciences, Deb ecen, Hunga y 4Depa men o De ma ology, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Deb ecen, Hunga y *These au ho s con ibu ed equally o he wo k #All co espondence should be add essed o: László Vi ág M.D. D.Sc. Depa men o Medical Chemis y Uni e si y o Deb ecen, MHSC Nagye dei k 98. H-4032 Deb ecen, Hunga y e-mail: l i [email protected] Tel: +36-52-412-345 Fax: +36-52-412-566 Key wo ds: melanoma, poly(ADP- ibose), poly(ADP- ibose) polyme ase, skin Running i le: PAR in he skin and in melanomas PAR in he skin and in melanomas His ol. His opa hol. 2 2 Summa y Cu aneous melanoma (CM) and u eal melanoma (UM) ep esen he mos agg essi e pigmen cell umo ypes. Ou in es iga ion examined he signaling molecule poly(ADP- ibose) (PAR) in CM and UM. We ha e demons a ed PAR in ke a inocy es, sebocy es, hai ollicles, endo helial cells and in subcu aneous adipocy es in he no mal skin indica ing ha PAR may egula e physiological unc ions in hese cell ypes. Fu he mo e, CM cells we e PAR posi i e and umo in asion le el/ hickness o CM co ela ed wi h he PAR con en o he cell nuclei, wi h highe Cla k and B eslow indices and AJCC sco es associa ing wi h highe PAR con en . This co ela ion was especially ma ked in he samples o emale pa ien s. In UM umo s (n=12) a sligh o e all and s ong pe i ascula PAR s aining was obse ed wi h conside able indi idual a ia ions. In iew o ecen success ul clinical ials wi h PARP inhibi o s as adju an chemo he apeu ic agen s, ou esul s sugges ha melanomas may display di e en ial sensi i i y owa ds his no el he apeu ic modali y which should be conside ed o he selec ion o pa ien s. PAR in he skin and in melanomas His ol. His opa hol. 3 3 In oduc ion Cu aneous melanoma (CM) is he mos agg essi e o m o skin cance among he Caucasian popula ion. I is he six h mos common cance and i s incidence is on he ise in wes e n popula ions. In he Uni ed S a es an es ima ed 68,720 new cases (39,080 men and 29,640 women) we e epo ed in 2009 causing 8650 dea hs (5550 men and 3100 women). The numbe o o al body acqui ed melanocy ic ne i and he occu ence o p e ious epi helial skin cance ep esen majo isk ac o s o he de elopmen o CM (Lawson e al., 1994; Ma ghoob e al., 1996; Jackson e al., 2000; Bakos e al., 2002). Sola damage is he majo en i onmen al causal ac o in all skin cance s, and in e mi en in ense exposu es o sunligh and/o se e e sunbu n especially du ing childhood (Hedges & Sc i en, 2008) a e also impo an en i onmen al isk ac o s o CM. The p ognosis o melanoma depends on he s age a which i is de ec ed as pa ien s who a e diagnosed ea ly and ea ed su gically wi h excision o he umo s ha e he highes chance o a comple e cu e. Un esec able o ad anced me as a ic diseases, howe e , ha e a poo p ognosis (Ga be e al., 1995; Ahmed 1997). The apeu ic s a egies o comba CM include chemo he apy, bio-chemo he apy, immune adju an s, cance -speci ic accines, cy okines, monoclonal an ibodies and speci ic immunos imulan s (Gue y & Schuch e , 1992, Schadendo 2002). Ano he pigmen cell-de i ed malignancy, u eal melanoma is he mos common adul p ima y in aocula umo (Sa o e al., 2008) howe e i appea s o be unlinked o pho odamage (Singh e al., 2004). U eal melanoma can be classi ied as spindle-A, spindle-B, mixed- ype and epi heloid melanomas wi h he o de o deg ada ion o he p ognosis ma ked by inc easing p opo ion o mi o ic cells, me as a ic capaci y and ex aocula ex ension. Possible ea men s ange om local lase pho ocoagula ion, adia ion he apy o enuclea ion dependen on he size o he melanoma. PAR in he skin and in melanomas His ol. His opa hol. 4 4 Inhibi ion o poly(ADP- ibose) polyme ases (PARPs) ecen ly eme ged as a no el he apeu ic modali y in he ea men o ce ain ypes o cance . F om he 17 membe PARP enzyme amily, he nuclea enzymes PARP-1 and PARP-2 a e DNA nick senso s (De Mu cia & Menissie de Mu cia, 1994; Ame e al., 2004,). Thei binding o damaged DNA induces enzyme ac i a ion whe eby PARPs clea e NAD+ o nico inamide and ADP- ibose and polyme ize he la e o o m b anched poly(ADP- ibose) (PAR) polyme (Sch eibe e al., 2006). PARyla ion egula es many p ocesses including ch oma in o ganiza ion, eplica ion, ansc ip ion, me abolism and cell dea h (Vi ag & Szabo, 2002; E delyi e al., 2005). PAR syn hesis is in ol ed in he ini ia ion o he DNA damage esponse acili a ing DNA base excision epai (Sch eibe e al., 2006; Hassa & Ho ige , 2008). PARP inhibi o s we e p oposed o be use ul as adju an he apeu ic agen s in cance pa ien s including melanoma pa ien s ecei ing chemo he apy o i adia ion ( Ten o i e al., 2003; Kaspe e al., 2007; Plumme e al., 2008; Chalme s, 2009). The exp ession o PARP-1 has been in es iga ed in CM (S aibano e al., 2005), howe e he signaling molecule PAR i sel was no de ec ed. PARP-1 is usually exp essed abundan ly in mos cell ypes and i s unc ion is egula ed p ima ily a he le el o ac i i y. PARP ac i i y is a ec ed by DNA b eaks, special DNA s uc u es, phospho yla ion and ace yla ion. Thus PAR le els do no necessa ily co ela e wi h p o ein le el o PARP-1 o o he PARP enzymes. He e we se ou o in es iga e he di e ences in PAR con en in a ious ypes o cu aneous and u eal melanomas. Ma e ials and me hods Tissue samples S udies in ol ing human issue samples we e conduc ed in acco dance wi h he decla a ion o Helsinki and we e app o ed by he E hical Commi ees o he Medical and PAR in he skin and in melanomas His ol. His opa hol. 5 5 Heal h Science Cen e o he Uni e si y o Deb ecen and Semmelweis Uni e si y ( o cu aneous and u eal melanoma samples, espec i ely). Re ospec i e analysis was pe o med on o malin- ixed, pa a in embedded ou ine his ology sec ions om he a chi e o he Depa men o De ma ology (Uni e si y o Deb ecen, Hunga y). Melanoma pa ien s (age 10-92 yea s) ea ed be ween 2002-2007 we e selec ed. Cla k s age I-V p ima y melanomas (B eslow 0,06-11,00 mm, T1a-4b) and cu aneous melanoma me as ases we e analyzed. Specimens we e classi ied by his ology as supe icial sp eading melanoma (17 subjec s), len igo melanoma (17 subjec s), nodula melanoma (17 subjec s), skin me as asis (15 subjec s). Heal hy skin (a ound melanocy ic nae i) and melanocy ic nae i we e used as con ols. U eal melanoma samples used in his s udy we e om pa ien s unde going enuclea ion due o UM a he Depa men o Oph halmology (Semmelweis Uni e si y, Budapes , Hunga y) be ween 2005 and 2006. We analyzed 12 o malin ixed, pa a in embedded umo s (n=12; 5 spindle, 3 epi heloid and 4 mixed). Ma e ials The an i-poly(ADP- ibose) an ibody (clone 10H) was pu chased om Alexis Biochemicals, (Lausanne, Swi ze land) (E delyi e al., 2009). The Vec o Eli e ABC ki (Vec o Labo a o ies, Bu lingame, CA, USA) was used o he immunos aining p ocedu e. Unless s a ed o he wise, all ma e ials we e ob ained om Sigma-Ald ich. Immunohis ochemis y The immunohis ochemical localiza ion o PAR was pe o med as ollows. A e depa a iniza ion, 5 m sec ions we e ea ed wi h H2O2 [3% ( / ) in me hanol] o 20 min in o de o block endogenous pe oxidase ac i i y. A e washing in PBS o 5 min, sec ions we e PAR in he skin and in melanomas His ol. His opa hol. 6 6 subjec ed o an igen e ie al by hea ing o 5 min in a p essu e cooke in sodium ci a e bu e (0.01 M, pH 6.0). Sec ions we e insed in dis illed wa e and washed o 5 min in PBS ollowed by incuba ion wi h a mix u e o 1% (w/ ) bo ine se um albumin and 1 % ( / ) ho se se um a oom empe a u e o 20 min, o p e en non-speci ic eac ions. Subsequen ly, sec ions we e incuba ed o e nigh a 4⁰C wi h an i-PAR monoclonal an ibody dilu ed in blocking solu ion (1% BSA, ho se se um). A e washing wi h PBS (3x10 min), sec ions we e u he incuba ed o 45 min a oom empe a u e wi h bio inyla ed ho se an i-mouse IgG seconda y an ibodies (p o ided in he Vec o ki and used a 1:600 dilu ion in blocking solu ion). The sec ions we e hen washed (3 × 10 min) wi h PBS, and ea ed wi h 2% a idin– bio in–pe oxidase complex (ABC) eagen o 30 min. A e wa ds, sec ions we e washed wi h PBS (3×10 min) and eac ed wi h Ni-DAB subs a e (1,6 mM 3,3′-diaminobenzidine e achlo ide, 140 mM NaCl, 90 mM NiSO4, 100 mM Na-ace a e, 3 mM H2O2, pH 6.6) o 4 minu es. A e insing sec ions in 0.1M TBS (pH 7,2), he colo was enhanced by incuba ing he sec ions o 3 min in 0.5% cobal chlo ide (in 0.1 M TBS, pH 7,2). A e insing in dis illed wa e , sec ions we e coun e s ained wi h Ch omo op 2R (Ch oma, S u ga , Ge many) solu ion (500 mg/l Ch omo op 2R, 0.005% ( / ) ace ic acid). Nega i e immunohis ochemical con ols (iso ype con ol an ibody) we e included in each s aining un. Ini ially, PAR de ec ion was pe o med a e emo al o melanin om he sec ions bu he p esence o melanin did no appea o in e e e wi h he immunode ec ion, he e o e his was omi ed om he inal p ocedu e. Sco ing and s a is ical analysis Nuclea s aining was sco ed semiquan i a i ely by wo expe ienced independen esea che s on an a bi a y scale as ollows: 0 (nega i e), 1 (low), 2 (mode a e), 3 (pa ly low PAR in he skin and in melanomas His ol. His opa hol. 7 7 o mode a e, pa ly s ong), 4 (s ong). Immunos ained slides we e compa ed o H&E s ained coun e pa s in o de o ensu e ha melanocy es we e sco ed. PAR s aining sco es o he wo independen in es iga o s we e a e aged and hen co ela ed wi h B eslow index, Cla k s age and AJCC sco es using G aphpad and SPSS17 so wa es. The Spea man’s coe icien o co ela ion was calcula ed and i s signi icance was de e mined a p<0.05. Resul s PAR o ma ion in heal hy skin and CM In no mal skin, PAR o ma ion was obse ed in a ious a eas (Figu e 1, A-G). Sebocy es and hai ollicle cells, epide mal ke a inocy es, endo helial cells and subcu aneous adipocy es all demons a ed posi i i y o PAR. In cu aneous melanoma sec ions we obse ed nuclea PAR s aining in he umo cells. Howe e , he in ensi y o he s aining was highly a iable (Figu e 1, H, I, J and Table 1). The s aining in ensi y co ela ed wi h B eslow index, Cla k s aging and AJCC sco es o he melanomas (Figu e 2). The Spea man’s ank coe icien s o he co ela ion be ween he PAR signal and B eslow index, Cla k s age and AJCC sco es o all samples we e =0.4125 (p=0.0025), = 0.3257 (p=0.0197) and =0.3657 (p=0.0083), espec i ely. In e es ingly, his co ela ion was mo e p onounced in he case o emale pa ien s: Spea man’s ank co ela ion coe icien s and hei s a is ical signi icance alues o he emale samples we e (in he same o de as abo e) =0.6678 (p=0.0004), =0.5584 (p=0.0046), =0,5486 (p=0.0055) and o he male pa ien s he alues we e =0.3227 (p= 0.1006), =0.2283 (p=0.2520) and =0.3575 (p=0.067). PAR in he skin and in melanomas His ol. His opa hol. 8 8 Speci ici y o he PAR s aining was u he con i med by s aining WM35 human melanoma cells (Fig 3A), ha had been ea ed wi h H2O2 (5 min, 400 μM) in he absence (Fig 3B) o p esence o he PARP inhibi o PJ34 (10 μM) (Fig 3C). PAR o ma ion in u eal melanoma In he una ec ed (no mal) a ea o he eye, nuclea PAR s aining was de ec ed in he nuclea laye s o he e ina (Fig 4) and a ound blood essels in he cho oid (A). By compa ison, PAR s aining a ied in loca ion wi hin he umo s (Fig 4B and C). In some samples, s aining was mos in ense a ound he blood essels (Fig 4D). Al hough we obse ed clea di e ences in PAR s aining be ween indi idual umo s, we we e unable o co ela e PAR immunoposi i i y wi h he g ade o he umo s due o he low numbe o cases in he s udy. Discussion Ou p esen s udy p o ides s ong e idence ha poly(ADP- ibose) is syn hesized in he no mal human skin and can also be de ec ed in CM and UM samples. In no mal skin, PAR posi i e cells we e ound in he epide mis. Ke a inocy es ha ep esen he majo i y o cells in he epide mis ha e al eady been desc ibed as exp essing PARP-1 (Ross e al., 1998; Szabo e al., 2001). Mo eo e , se e al g oups ha e epo ed a dominan exp ession o PARP-1 in cul u ed p ima y ke a inocy es o HaCaT cells ( Malanga & Al haus, 1994; Szabo e al., 2001; Bakondi e al., 2002; Pache nik e al., 2002; Bakondi e al., 2003; Bakondi e al., 2004; Cals-G ie son & O me od, 2004). In ke a inocy es, PARP-1 ac i a ion has been linked o in lamma o y skin diseases and sunbu n (Szabo e al., 2001; Fa kas e al., 2002; Bakondi e al., 2004; Bai e al., 2009). PARP1 has also been shown o egula e he exp ession o p oin lamma o y cy okines and chemokines (IL-1, TNFa, MIP-1a, PAR in he skin and in melanomas His ol. His opa hol. 9 9 MIP-2, MCP-1, e c.) (Szabo e al., 2001; Bakondi e al., 2003; Cals-G ie son & O me od, 2004,; Bai e al., 2009) and oxida i e s ess–induced cell dea h (Bakondi e al., 2003; Bakondi e al., 2004). Ou cu en s udy indica es ha PAR may also se e as a signaling molecule in ke a inocy es as he polyme could be de ec ed in ke a inocy es in he heal hy skin. Howe e , whe he PAR syn hesis in ke a inocy es is induced by DNA b eaks (e.g. gene a ed by opoisome ases) o by DNA b eak-independen p ocesses, equi es u he in es iga ion. In ense PAR s aining was ound in hai ollicle cells. This may be due o he apid cycling o cells as PARyla ion has been implica ed in he egula ion o p oli e a ion (Vi ag & Szabo, 2002). We also de ec ed PAR polyme in sebocy es and adipocy es sugges ing a new ole o PAR in hese lipid accumula ing cell ypes. The ole o PARP-1 in adipocy es has al eady been p oposed (Janssen & Hilz, 1989; Hsu & Yen, 2006), howe e , i s p esence in ma u e adipocy es has no ye been epo ed. PARP-1 has been shown o in e ac wi h he e inoid X- ecep o (RXR) in he RXR – pe oxisome p oli e a o ac i a ed ecep o  (PPAR) nuclea ecep o he e odime (Miyamo o e al., 1999) ha con ols sebocy e lipid accumula ion du ing sebocy e di e en ia ion ( Rosen ield e al., 1999; Kim e al., 2001). Recen da a sugges s ha ec ui men o PARP-1 o ac i e nuclea ecep o s is ini ia ed by DNA s and b eaks du ing ecep o ac i a ion (Ju e al., 2006) hus possibly accoun ing o he p esence o PAR in hese cells. PARP-1 exp ession has p e iously been de ec ed in human melanomas wi h co ela ion es ablished be ween he in ensi y o exp ession and he clinical s age o he umo (S aibano e al., 2005). Howe e , PAR con en may e lec he in ensi y o cellula PAR me abolism be e han PARP-1 exp ession. The e o e, ou indings ha PAR con en co ela es wi h he B eslow index, Cla k s age and AJCC sco e o he umo may ha e he apeu ic implica ions in pa ien s ea ed wi h PARP inhibi o s as adju an chemo he apeu ic agen s. 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PAR in he skin and in melanomas His ol His opa hol 19 19 Age Sex B eslow Cla k AJCC His ology PAR Age Sex B eslow Cla k AJCC His ology PAR 62 M 0,45 3 1 SSM 1 44 M 2,5 5 1,5 NM 1,5 33 M 0,24 3 1 SSM 1 53 M 4,5 4 2 NM 2 69 M 1,12 3 1 SSM 1,5 39 F 2 3 2 NM 2,5 44 F 0,48 3 4 SSM 1 68 F 6,5 4 4 NM 3 44 F 6 3 2 SSM 3 65 F 5,5 5 4 NM 3 38 F 0,12 2 1 SSM 2 42 F 2 4 2 NM 3 73 F 1,75 4 2 SSM 2 51 F 3 2,5 3 NM 2 63 F 1,1 3 2 SSM 3 51 F 2,5 4 3 NM 2 40 F 7 4 4 SSM 3 64 F 2,45 3 3 NM 3 69 F 2 4 2 SSM 2 75 F 11 5 4 NM 4 58 M 0,61 3 1 SSM 1 35 F 8,5 5 4 NM 2,5 44 M 0,88 3 1 SSM 2 55 M 0,65 3 1 NM 1,5 63 M 0,25 2 1 SSM 2 69 M 1,61 4 2 NM 3,5 51 M 0,65 3 1 SSM 3 40 M 4 4 3 NM 3 38 M 0,55 3 1 SSM 3 66 M 3,8 4 3 NM 4 50 M 0,31 2 1 SSM 1 56 M 1 3 1 NM 4 45 M 1,12 3 2 SSM 3,5 44 M 2,25 4 3 NM 4 67 M 0,12 2 1 LM 1 83 M MET 4 71 M 0,06 2 1 LM 2 66 M MET 3 79 F 0,42 3 1 LM 1 10 M MET 2,5 66 F 0,2 1,5 1 LM 2 83 M MET 3 81 F 0,07 2 1 LM 2 92 F MET 3 80 F 0,18 2 1 LM 2 45 F MET 2 85 F 0,3 2 1 LM 2,5 46 F MET 2 73 F 0,22 3 1 LM 2 60 F MET 4 65 F 0,28 2 1 LM 1 64 F MET 3,5 45 F 0,2 1 1 LM 1 10 F MET 2 69 M 0 1 1 LM 3 52 M MET 3 82 M 0 1 1 LM 3 62 M MET 4 70 M 0,85 3 1 LM 4 68 M MET 3 77 M 1,4 3 2 LM 2 77 M MET 3,5 75 M 0,3 2 1 LM 2 51 M MET 2 71 M 0,43 2 1 LM 3 68 M 0,56 3 1 LM 4 Table 1.  PAR in he skin and in melanomas His ol His opa hol 20 20 Age His opa hologic cell ype Ex aocula ex ension PAR s aining 64 spindle no 1 55 mixed no 2 55 epi heloid scle al in il a ion 2 71 epi heloid op icus in il a ion 2 76 spindle no 4 46 spindle no 4 53 mixed scle al in il a ion 3 46 mixed o ex ein 3 39 spindle no 3 81 spindle scle al in il a ion 3 56 epi heloid no 3 54 mixed no 2 Table 2.