Substantial expression of luteinizing hormone-releasing hormone (LHRH) receptor type I in human uveal melanoma

Onco a ge 2013; 4:1721 www.impac jou nals.com/onco a ge www.impac jou nals.com/onco a ge / Onco a ge , Oc obe , Vol.4, No 10 Subs an ial exp ession o lu einizing ho mone- eleasing ho mone (LHRH) ecep o ype I in human u eal melanoma And ea T eszl1,*, Zi a S eibe 2,*, And ew V. Schally3,4 , No man L Block3,4 , Balazs Dezso5, Gabo Olah1, Be nade Rozsa1, Kla a Fodo 1, A min Buglyo1, Janos Ga di6, And as Be a2 and Gabo Halmos1,3 1 Depa men o Biopha macy, Uni e si y o Deb ecen, Medical and Heal h Science Cen e , Deb ecen, Hunga y 2 Depa men o Oph halmology, Uni e si y o Deb ecen, Medical and Heal h Science Cen e , Deb ecen, Hunga y 3 Ve e ans A ai s Medical Cen e Miami, FL, Sou h Flo ida VA Founda ion o Resea ch and Educa ion, Miami, FL and Depa men o Pa hology, Uni e si y o Miami, Mille School o Medicine, Miami, FL, USA 4 Di isions o Hema ology/Oncology and Endoc inology, Depa men o Medicine, Uni e si y o Miami, Mille School o Medicine, Miami, FL, USA 5 Depa men o Pa hology, Uni e si y o Deb ecen, Medical and Heal h Science Cen e , Deb ecen, Hunga y 6 Depa men o Endoc inology, Uni e si y o Szeged, Facul y o Medicine, Szeged, Hunga y * These au ho s con ibu ed equally o his wo k. Co espondence o: Gabo Halmos, email: [email p o ec ed] Co espondence o: And ew V. Schally, email: [email p o ec ed] Keywo ds: u eal melanoma, lu einizing ho mone- eleasing ho mone (LHRH) ecep o Recei ed: Sep embe 5, 2013 Accep ed: Sep embe 8, 2013 Published: Sep embe 10, 2013 This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. ABSTRACT: U eal melanoma is he mos common p ima y in aocula malignancy in adul s, wi h a e y high mo ali y a e due o equen li e me as ases. Consequen ly, he he apy o u eal melanoma emains a majo clinical challenge and new ea men app oaches a e needed. Fo imp o ing diagnosis and designing a a ional and e ec i e he apy, i is essen ial o elucida e molecula cha ac e is ics o his malignancy. The aim o his s udy he e o e was o e alua e as a po en ial he apeu ic a ge he exp ession o lu einizing ho mone- eleasing ho mone (LHRH) ecep o in human u eal melanoma. The exp ession o LHRH ligand and LHRH ecep o ansc ip o ms was s udied in 39 human u eal melanoma specimens by RT-PCR using gene speci ic p ime s. The binding cha ach e is ics o ecep o s o LHRH on 10 samples we e de e mined by ligand compe i ion assays. The p esence o LHRH ecep o p o ein was u he e alua ed by immunohis ochemis y. The exp ession o mRNA o ype I LHRH ecep o was de ec ed in 18 o 39 (46%) o issue specimens. mRNA o LHRH-I ligand could be de ec ed in 27 o 39 (69%) o he samples. Se en o 10 samples in es iga ed showed high a ini y LHRH-I ecep o s. The speci ic p esence o ull leng h LHRH ecep o p o ein was u he con i med by immunohis ochemis y. A high pe cen age o u eal melanomas exp ess mRNA and p o ein o ype-I LHRH ecep o s. Ou esul s suppo he me i o u he in es iga ion o LHRH ecep o s in human oph halmological umo s. Since di e se analogs o LHRH a e in clinical ials o a e al eady used o he ea men o a ious cance s, hese analogs could be conside ed o he LHRH ecep o -based ea men o u eal melanoma. INTRODUCTION Al hough u eal melanoma is e y a e, i is he mos common p ima y in aocula malignancy in adul s. I s incidence in he Wes e n wo ld seems o be ela i ely s able wi h abou 7 new cases pe yea pe 1 million indi idual. Abou hal o he pa ien s al eady ha e me as a ic disease by he ime o diagnosis. The ou come Onco a ge 2013; 4:1722 www.impac jou nals.com/onco a ge o hese is almos in a iably a al and dea h usually occu s wi hin a yea o he onse o sys emic symp oms. [1,2] T ea men by sys emic o in a-hepa ic chemo he apy o pa ial hepa ec omy only a ely p olongs he su i al [3], emphasizing he need o de elop mo e e icacious he apies. Recen p og ess in ou unde s anding o he molecula p ocesses unde lying u eal melanoma should enable us o ad ance he diagnosis, p ognosis and ea men o his malignancy. The disco e y o speci ic ecep o s o pep ide ho mones on cance cells has led o he de elopmen o cy o oxic and adiolabeled ho mone analogs ha a e use ul o umo localiza ion and a ge ed he apy. Va ious p eclinical s udies ha e shown ha chemo he apy based on cy o oxic pep ide conjuga es a ge ed o ecep o s on umo s can imp o e he e ec i eness o ea men and educe gene al side e ec s. [4] The p esence o di e en iso o ms o Lu einizing Ho mone-Releasing Ho mone (LHRH) also known as Gonado opin Ho mone-Releasing Ho mone has been iden i ied in e eb a es. LHRH is he p ima y link be ween he b ain and he pi ui a y in he egula ion o gonadal unc ion and plays a pi o al ole in e eb a e ep oduc ion. The ac ions o LHRH a e media ed by high a ini y ecep o s o LHRH. The disco e y o LHRH has had a majo impac in medicine and has led o a a ie y o clinical uses o LHRH analogs in oncology and gynecology. [5] Recen ly, i has been shown ha a ious cance cell lines, including cu aneous melanoma, xenog a ed in o nude mice, can be inhibi ed by he a ge ed cy o oxic LHRH analog AN-152 (AEZS-108). [6] Since bo h cu aneous melanoma and u eal melanoma ha e he same neu oec ode mal o igin, bu he p esence o LHRH ecep o s has ne e been s udied in u eal melanoma, we in es iga ed he exp ession o mRNAs o LHRH-I ligand and o ype I LHRH ecep o in specimens o human u eal melanoma. The p esence and binding cha ac e is ics o LHRH ecep o p o ein we e also examined. RESULTS Exp ession o human ype-I LHRH ecep o s in human u eal melanoma Ou specimens o u eal melanoma issue consis ed o 11 epi helioid, 21 spindle and 7 mixed cell ype umo s. The umo hickness ange was 6-12.9 mm acco ding o ul asonog aphy. The umo basal diame e s as measu ed wi h ul asonog aphy, anged om 9-19 mm. I he umo hickness was mo e han 8 mm and/o he la ges umo diame e was mo e han 13 mm, we enuclea ed he eye wi hou p io ea men . In hose cases whe e he hickness was less han 8 mm o he basal diame e was less han 13 mm, bu he umo was g owing in spi e o he p e ious anspupilla y he mo he apy and/o Ru henium-106 plaque b achy he apy, enuclea ion was pe o med. Based on ou ecen knowledge, ype I LHRH ecep o has wo splice a ian s, bu only he ull leng h ecep o is unc ional. Ou p ime se o LHRH ecep o was designed o speci ically ampli y he mRNA o he ull leng h ecep o , bu o gi e no signals o he splice a ian s. We used LHRH ecep o p ime s encompassing he open eading ame om exon 2 o exon 3, o e lapping he missing pa in he abo e men ioned wo iso o ms. The p edic ed size o he PCR ampli ied cDNA o ype I LHRH ecep o was 241 bp. Fou y six pe cen o ou samples exp essed ecep o s o ype I LHRH ecep o (Fig. 1., Table 1.). Among epi helioid ype u eal melanomas, 6 o 11 (55 %) we e ound o be posi i e o he exp ession o LHRH ecep o while spindle ype melanomas included 10 o 21 (48%) posi i e samples. In he mixed cell ype g oup (con aining bo h epi helioid and spindle cells, wi h no dominan pa e n), 2 o 7 (29%) o he umo s exp essed ype I LHRH ecep o . The p esence o ull leng h LHRH-I ecep o was con i med by immunohis ochemis y and co ela ed well wi h he indings by RT-PCR (Fig. 2.). Among he specimens o RT-PCR posi i e u eal melanoma, he majo i y s ained posi i e o LHRH ecep o s which could be de ec ed in he o m o ed g anules. The p esence o speci ic LHRH binding si es and cha ac e is ics o binding o [125I][D-T p6]LHRH o memb ane ecep o s on human u eal melanoma issue was de e mined using ligand compe i ion assays. O he 10 specimens examined, 7 showed LHRH ecep o binding (Table 2.). Analyses o he ypical displacemen o adiolabeled [D-T p6]LHRH by he same unlabeled pep ide e ealed ha he one-si e model p o ided he bes i , indica ing he p esence o one class o high-a ini y LHRH ecep o s in c ude memb anes de i ed om human u eal melanoma specimens. The compu e ized nonlinea cu e- i ing and he Sca cha d plo analyses o he binding da a in he 7 ecep o -posi i e umo specimens indica ed ha he single class o binding si es had a mean dissocia ion Figu e 1: mRNA exp ession o LHRH-I (le panel) and i s ype I ecep o ( igh panel) in he same issue specimen se . L: 50 bp DNA-ladde (Fe men as); +: posi i e con ol (human pi ui a y); -: no empla e con ol; No. 1-5: ep esen a i e human u eal melanoma issues. Onco a ge 2013; 4:1723 www.impac jou nals.com/onco a ge Table 1: Clinicopa hological cha ac e is ics and RT-PCR esul s o enuclea ed u eal melanoma samples Numbe o pa ien Age a enuclea ion Sex His ology Ea lie he apy Eye LHRH-R LHRH ligand 1. 64 M Spindle - L + + 2. 39 M Epi helioid B achy he apy R +- 3. 70 M Spindle - R - + 4. 80 F Epi helioid - R +- 5. 47 M Epi helioid - L - - 6. 65 M Spindle - R +- 7. 76 M Spindle - R + + 8. 75 F Spindle - L - + 9. 84 F Spindle B achy he apy L + + 10. 39 M Mixed - R - + 11. 35 M Spindle - L - - 12. 30 F Spindle - L + + 13. 44 F Spinde - L - + 14. 68 F Spindle - L - + 15. 76 M Spindle - R + + 16. 79 F Mixed - L - + 17. 79 F Epi helioid - R + + 18. 67 M Epi helioid B achy he apy, TTT L+ + 19. 60 M Epi helioid B achy he apy (3x) R + + 20. 51 M Spindle - L - + 21. 66 M Epi helioid - R + + 22. 72 F Epi helioid - L - + 23. 76 M Spindle - L + + 24. 55 M Spindle - L - + 25. 54 M Epi helioid - R - + 26. 75 F Spindle - L + + 27. 64 F Epi helioid - R - + 28. 61 M Spindle - L - + 29. 50 F Spindle - R +- 30. 76 M Mixed - L + + 31. 38 M Spindle - R - + 32. 79 F Mixed - R - - 33. 53 M Epi helioid - R - + 34. 52 M Mixed - L - + 35. 45 M Mixed - R +- 36. 70 M Spindle - R +- 37. 43 M Spindle - L - - 38. 53 M Mixed - L - - 39. 51 M Spindle B achy he apy (3x) L - - L: le , R: igh , TTT: T anspupilla y The mo he apy Onco a ge 2013; 4:1724 www.impac jou nals.com/onco a ge cons an (Kd) o 3.69 nM ( ange, 1.35 o 6.36 nM) wi h a mean maximal binding capaci y (Bmax) o 384.5 mol/ mg o memb ane p o ein ( ange, 251.5 o 511.6 mol/mg p o ein). Biochemical pa ame e s essen ial o es ablish he iden i y o speci ic binding si es we e also de e mined. Thus he binding o [125I][D-T p6] LHRH was ound o be e e sible, ime- and empe a u e-dependen , and linea wi h p o ein concen a ion in he human u eal melanoma specimens examined (da a no shown). The speci ici y o LHRH binding was demons a ed by compe i i e binding expe imen s using se e al pep ides s uc u ally ela ed o un ela ed o LHRH (da a no shown). The exp ession o mRNA o LHRH ecep o s was accompanied by ligand binding in all samples examined. Th ee o 10 umo specimens did no exhibi mRNA exp ession o ype I LHRH ecep o s, o show ligand binding; con e sely, all ecep o -posi i e specimens exp essed a de ec able amoun o he ecep o gene (Table 2.). The e was no e iden co ela ion be ween ecep o binding cha ac e is ics o mRNA exp ession and clinical and pa hological indings. Exp ession o mRNA o human ype I LHRH in human u eal melanoma In addi ion o he exp ession o ype-I LHRH ecep o , we also s udied he p esence o LHRH ligand in ou u eal melanoma samples. The majo i y o u eal melanomas (27 o 39, 69%) showed ma ked exp ession o mRNA o LHRH-I ligand (Table 1.). The expec ed size o he PCR p oduc o 245 bp could be de ec ed in 8 o 11 (73% ) o epi helioid and 15 o 21 (71%) o spindle ype umo s. In he mixed umo ype, 4 o 7 (57%) exp essed LHRH-I (Fig. 1.). In 12 umo s (31%), bo h LHRH-I ligand and ecep o we e exp essed. In 6 samples, only ype I LHRH ecep o s we e p esen , while in 15 melanomas only LHRH-I ligand was p esen (Tabel 1.). S a is ical analysis showed an associa ion be ween age and LHRH ecep o mRNA and ligand co-exp ession (p=0.0407): a 10-yea inc ease o age mean an es ima ed 87% inc ease in he odds o co-exp ession (OR=1.867, 95%CI 1.027 o 3.393). O he wise, he e we e no co ela ions be ween LHRH-I ligand o ecep o mRNA exp ession and umo sub ype o clinical pa ame e s. The clinicopa hological cha ac e is ics o pa ien s wi h u eal melanoma and he esul o mRNA exp ession analysis a e summa ized in Table 1. Figu e 2: Exp ession o LHRH ecep o p o ein in enuclea ed human u eal melanoma issue samples demons a ed by immunope oxidase s aining. A, Hema oxylin-eosin s ained sec ion o a ep esen a i e sample shows melanin-p oducing neoplas ic cells wi h spindle and epi helioid pa e n. a-d, images o ep esen a i e samples immunos ained o ype I LHRH ecep o ; B, umo sample wi hou de ec able mRNA o ype I LHRH ecep o also e ealed no iden i iable LHRH ecep o p o ein using IHC s aining; C, ep esen a i e issue sec ion shows mild posi i i y o ype I LHRH ecep o ( ain ed cy oplasms o umo cells). Inse is a nega i e con ol o he s aining-speci ici y (see Me hods); D, ep esen a i e umo sample exhibi s in ense exp ession o ype I LHRH ecep o in nea ly 100% o umo cells (in ense ed cy oplasmic s aining) which co ela ed wi h he co esponding mRNA le els. O iginal magni ica ions o all images: 40x. Images B-D a e immunope oxidase s ained sec ions wi h hema oxylin nuclea coun e s aining. Table 2: Exp ession o mRNA and binding cha ac e is ics o ype I ecep o s o LHRH (LHRH-R) in 10 human u eal melanoma specimens. Pa ien No. LHRH-R mRNA Bmax ( mol/mg p o ein) Kd (nM) 4. +294.6 1.35 6. +398.7 1.37 9. +264.7 3.73 11. - - - 12. +495.7 6.03 15. +251.5 4.81 20. - - - 24. - - - 30. +511.6 6.36 35. +474.7 2.18 Onco a ge 2013; 4:1725 www.impac jou nals.com/onco a ge DISCUSSION Pa ien s wi h u eal melanoma ace a dismal p ognosis as e en ually abou 45% o hem die o me as asis, ega dless o he ac ha he umo is mos o en diagnosed and locally cu ed be o e any signs o clinical dissemina ed disease appea . [7] This ga e ise o he heo y ha mic ome as ases a e al eady p esen ea ly in he disease p ocess, bu emain do man o yea s be o e a clinically de ec able mac ome as asis de elops. [8] Mos small and medium-sized u eal melanomas a e cu en ly managed by di e en o ms o adio he apy and local esec ion. Howe e , i emains unclea wha e ec his ea men has on pa ien su i al. Sys emic ea men op ions o u eal melanoma a e e y limi ed. Adju an sys emic he apy is mainly used in pa ien s a high- isk o me as asis o in pa ien s who ha e al eady de eloped me as asis, bu he esponse a es o classical chemo he apeu ic agen s emain as low as 7%–25%. [9] Despi e he imp o emen s in diagnosis and he apy o p ima y u eal melanoma in he las 20–30 yea s, he e has been no signi ican dec ease in me as asis- ela ed dea hs. [10,11] Thus, he de elopmen o new he apeu ic modali ies is manda ed. The p esence o ecep o s o LHRH in a ious cance s and cance cell lines o igina ing om o gans o he han hose o he ep oduc i e sys em has been demons a ed by se e al au ho s. [6,12-15] I was sugges ed ha he signal- ansduc ion mechanisms media ed by ype I o LHRH ecep o a e di e en in he pi ui a y and in cance cells. [16,17] I appea s ha in cance cells LHRH analogs in e e e wi h mi ogenic signal ansduc ion o g ow h- ac o ecep o s and ela ed oncogene p oduc s associa ed wi h ac i i ies o y osine kinases. [17,18] I has been shown ha cu aneous melanomas also exp ess ecep o s o LHRH. [6,13] The ea men o melanoma cells by agonis s o LHRH o cy o oxic analogs o LHRH signi ican ly inhibi s cell p oli e a ion. [6,13] As u eal melanoma and melanoma o he skin a e bo h o neu al c es o igin and sha e ce ain gene ic cha ac e is ics, we analyzed he exp ession o LHRH ecep o s in u eal melanoma by RT-PCR, ligand compe i ion assay and immunohis ochemis y. As only he ull leng h ype I LHRH ecep o is unc ional, ou p ime se was designed o selec i ely ampli y ecep o mRNA encoding he ull leng h ype I LHRH ecep o bu no i s known splice a ian s. The an ibody used o immunohis ochemis y was also chosen o de ec ull leng h p o ein. In ou p esen s udy, we ound ha 47% o ou samples exp essed ype I ecep o s o LHRH. Fu he mo e, using ligand compe i ion assay we examined he binding o [ 125 I][D-T p 6 ]LHRH o memb ane p epa a ions o 10 u eal melanoma specimens. We ound ha 70% o he human u eal melanoma samples in es iga ed possessed speci ic ype I LHRH ecep o s wi h a mean Kd o 3.69 nM and wi h a mean Bmax o 384.5 mol/mg memb ane p o ein. I is also impo an o no e ha all ecep o -posi i e specimens exp essed a de ec able amoun o he ecep o gene. The ecep o p o ein encoded by mRNA o ype I LHRH ecep o s was also demons a ed by immunohis ochemis y in umo specimens. The high incidence o posi i i y o ype I LHRH ecep o in u eal melanoma sugges s ha his umo ype migh be a good candida e o he apy wi h LHRH analogs including he a ge ed cy o oxic pep ide, AN-152 (AEZS- 108). AN-152 is al eady in phase III clinical ials in women wi h endome ial and o a ian cance s [18-20] and in phase I/II ials in men wi h cas a ion esis an p os a e cance [21, Liu S, Schally AV, Do TB, Tsao-Wei DD, G oshen SG, Xiong S, Hawes D, Quinn DI, Tai YC, Block NL, Engel J, Pinski JK. A phase I/II ial o AN- 152, a a ge ed cy o oxic LHRH analog, in cas a ion- and axane- esis an p os a e cance . ASCO Annual Mee ing June 3-7, 2011, Chicago, Abs ac #74003] and pa ien s wi h u o helial ca cinoma [Fe nandez GL, Schally AV, Ko u-Sengul T, Me chan JR, Flo es AM, Jo da M, Da a R, Benede o PW, Singal R, Block NL, Engel J. A phase I/II ial o AEZS-108 in locally ad anced un esec able o me as a ic lu einizing ho mone- eleasing ho mone (LHRH) posi i e u o helial ca cinoma (UC) pa ien s who ailed pla inum based chemo he apy. ASCO Annual Mee ing June 3-7, 2011 Chicago, Abs ac # 83230]. Ta ge ed he apy wi h cy o oxic pep ide analogs consis ing o a pep ide molecule conjuga ed o a cy o oxic moie y such as doxo ubicin, should be mo e e ec i e and less oxic han con en ional sys emic chemo he apy [20,22]. Thei only side e ec appea s o be myelosupp ession caused by he occasional chemical clea age o he cy o oxic adical doxo ubicin. [4,5,18-20,22] The subs an ial exp ession o LHRH-I ligand (69%) and equen co-exp ession o i s ecep o (31%) in ou sample se may be indica i e o he p esence o an au oc ine/ pa ac ine egula o y sys em based on LHRH in u eal melanoma. The egula ion o se e al p o eins associa ed wi h cell p oli e a ion and cell mo ili y is media ed by ype I LHRH ecep o /LHRH-I sys em, sugges ing hei impo an ole in me as asis o ma ion. [23] Howe e , he ole o his high exp ession o LHRH ound by us is no clea . To he bes o ou knowledge, ou indings ep esen he i s iden i ica ion o LHRH and i s ecep o s in human u eal melanoma. Since he he apy o his malignancy is no adequa e, ou wo k may help o iden i y speci ic molecula a ge s o he p e en ion o me as asis o u he p oli e a ion o al eady dissemina ed me as ases. Ou indings ha a high pe cen age o human u eal melanoma specimens exp ess ecep o s o LHRH suppo he iew ha a ge ed cy o oxic LHRH analogs such as AN-152 could be used o an e ec i e ea men o u eal melanoma. Onco a ge 2013; 4:1726 www.impac jou nals.com/onco a ge METHODS Pa ien s and Tissue Samples Human u eal melanoma specimens we e ob ained om 39 pa ien s, 30-84 yea s o age a he ime o enuclea ion, a he Depa men o Oph halmology, Uni e si y o Deb ecen, Hunga y. No mal pi ui a y samples (an e io lobe) we e collec ed a au opsy a he Depa men o Pa hology, Uni e si y o Deb ecen and we e used as posi i e con ols. A e su gical emo al, selec ed po ions o he melanoma issues we e lash ozen and s o ed a -70°C. His opa hological examina ion o each specimen was unde aken o con i m he diagnosis. The local Ins i u ional E hics Commi ee app o ed he collec ion and use o hese specimens o he cu en s udy and in o med consen was ob ained om hese pa ien s. RNA isola ion, Re e se ansc ip ion and RT- PCR To al RNA was isola ed using AllP ep DNA/ RNA/P o ein Mini ki acco ding o he manu ac u e ’s ins uc ions (Qiagen, Hilden, Ge many). Two hund ed i y nanog ams o RNA om each sample we e e e se ansc ibed in o cDNA by Quan iTec Re e se T ansc ip ion ki (Qiagen) in a inal olume o 20 µl. Two p ime se s we e designed o e alua e he exp ession o ype I LHRH ecep o s (sense: 5’-GGTGGCATCAAGCATTTTAT-3’, an isense: 5’– ACATAGTAGGGAGTCCAGCAGACA-3’) and LHRH ligand (sense: 5’–GGCCTTATTCTACTGACTTGG-3’, an isense: 5’-TCTTCTGCCCAGTTTCCTCT-3’). As in e nal con ol, β-ac in housekeeping gene (sense: 5’-GGCATCCTCACCCTGAAGTA-3’, an isense 5’-GGGGTGTTGAAGGTCTCAAA-3’) was used. In all PCR eac ions, 1 µl o cDNA was ampli ied in a 25 µl solu ion con aining 1.5 mM MgCl2, 1x PCR bu e (Fe men as GmbH, S . Leon-Ro , Ge many), 0.3 mM o each deoxynucleo ide (P omega, Madison, WI), 1 uni o T ueS a Ho S a DNA polyme ase (Fe men as) and 0.25 µM o each p ime . Samples we e dena u ed o 3 min a 95°C, hen subjec ed o 40 cycles a 95°C o 45 s, 59°C o 30 s, hen 72°C o 1.5 min wi h a inal ex ension a 72°C o 10 min. Ten µl o each ampli ica ion eac ion was hen elec opho e ically sepa a ed on 1.5% aga ose gel, s ained wi h e hidium b omide, and isualized unde UV ligh . P epa a ion o memb anes and adioligand binding s udies P epa a ion o memb anes o ecep o s udies was pe o med as desc ibed p e iously. [24] B ie ly, he samples we e hawed, cleaned, and hen homogenized in 50 mM T is-HCl bu e (pH 7.4), supplemen ed wi h p o ease inhibi o s (0.25mM Phenylme hylsul onyl Fluo ide, 0.4% ( / ) Ap o inin and 2 µg/ml Peps a in A) using an Ul a-Tu ax issue homogenize (IKA Wo ks, Wilming on, NC) on ice. The homogena e was cen i uged a 500x g o 10 minu es a 4 ºC o emo e nuclea deb is and lipid laye . The supe na an con aining he c ude memb ane ac ion was ul acen i uged (Beckman L8-80 M) wice a 70,000x g o 50 minu es a 4 ºC a e esuspending in esh bu e . The inal pelle was esuspended in homogeniza ion bu e and s o ed a -80 ºC un il assayed. P o ein concen a ion was de e mined by he me hod o B ad o d using a Bio-Rad p o ein assay ki (Bio-Rad Labo a o ies, He cules, CA). Radio-iodina ed de i a i es o [D-T p6]LHRH we e p epa ed by he chlo amine-T me hod and pu i ied by e e se-phase HPLC in ou labo a o y. [24] LHRH ecep o binding assays we e ca ied ou as epo ed [24] using in i o ligand compe i ion assays based on binding o [125I][D-T p6]LHRH as adioligand o u eal melanoma memb ane ac ions. This adioligand has been well- cha ac e ized p e iously and shows high-a ini y binding o human and a pi ui a ies as well as human b eas , p os a e, and o he cance s. [4-6,22,24] In b ie , memb ane homogena es con aining 50-160 µg p o ein we e incuba ed in duplica e o iplica e wi h 60-80,000 cpm [125I][D-T p6] LHRH and inc easing concen a ions (10-12 - 10-6 M) o non adioac i e pep ides as compe i o s in a o al olume o 150 µl o binding bu e . A he end o he incuba ion, 125 µl aliquo s o suspension we e ans e ed on o he op o 1 ml o ice-cold binding bu e con aining 1.5% bo ine se um albumin in siliconized polyp opylene mic ocen i uge ubes (Sigma-Ald ich GmbH, Munich, Ge many). The ubes we e hen cen i uged a 12,000x g o 3 minu es a 4 ºC (Beckman J2-21M). Supe na an s we e aspi a ed and he bo oms o he ubes con aining he pelle we e cu o and coun ed in a gamma coun e (Mic omedic Sys em, Hun s ille, AL). P elimina y expe imen s we e pe o med wi h memb ane p o ein concen a ions anging om 20-250 µg/ ube in o de o de e mine he minimal amoun o p o ein equi ed o assess speci ic binding a a sa is ac o y le el. Ou wo k showed ha accu a e esul s can be ob ained o e a ange o 40-180 µg o memb ane p o ein in an incuba ion olume o 150 µl. Onco a ge 2013; 4:1727 www.impac jou nals.com/onco a ge Immunohis ochemis y (IHC) Fo malin- ixed pa a in-embedded issue samples om enuclea ion we e immunos ained as desc ibed ea lie . [24] B ie ly, ollowing an igen- e ie ing (pH: 6.0) and endogenous pe oxidase-block, 3 μm hick sec ions we e incuba ed wi h monoclonal an ibody o LHRH-RI (NCL-GnRHR A9E4; No ocas a Labo a o ies L d., UK) a oom empe a u e o 1 hou . A e insing 3 imes in phospha e-bu e ed saline (PBS; pH:7.4, 5 mins each), sec ions we e ea ed wi h an i-mouse IgG (Fab)2- coupled o ho se- adish-pe oxidase (HRP) o EnVision+- HRP de ec ion ki using amino-e hyl-ca basol (AEC; ed; Vec o Labs, UK) pe oxidase subs a e acco ding o he manu ac u e ’s ins uc ions. The use o ed ch omogenic subs a e a oided he colo -in e e ence be ween he b own melanin pigmen s o melanomas and he posi i e s aining o IHC. Human pi ui a y glands (an e io lobe) ob ained om au opsy we e used as posi i e con ols. Nega i e con ols in which p ima y an ibody was eplaced by no mal se um we e also included o each IHC- un. S a is ical analysis Va iables we e desc ibed using s anda d s a is ics. Associa ion be ween ca ego ical a iables we e assessed using Fishe ’s exac es s, while hose be ween con inuous explana o y a iables and bina y ou comes we e assessed using logis ic eg ession, and exp essed in e ms o odds a io (OR) and 95% con idence in e als (CI). ACKNOWLEDGEMENT This wo k was suppo ed by Zol an Magya y Pos doc o al Fellowship suppo ed by EEA G an s and No way G an s (A.T.), Hunga ian Scien i ic Resea ch Fund (OTKA) K 81596 (G.H.) and TAMOP 4.2.2.A- 11/1/KONV-2012-0025 p ojec (G.H.). The p ojec is co- inanced by he Eu opean Union and he Eu opean Social Fund. No po en ial con lic o in e es exis s o he au ho s. REFERENCES 1. Dama o B. Does ocula ea men o u eal melanoma in luence su i al? B J Cance . 2010;103:285-290. 2. Eskelin S, Py hönen S, Hahka-Kemppinen M, Tuomaala S, Ki elä T. A p ognos ic model and s aging o me as a ic u eal melanoma. Cance . 2003;97:465–475. 3. Augsbu ge JJ, Co ea ZM, Shaikh AH. E ec i eness o ea men s o me as a ic u eal melanoma. 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