Immunohistochemical detection of metallothionein

33 he eby in luencing he unc ion o nume ous enzymes and ansc ip ion ac o s ha con - ol cell p oli e a ion, apop osis and signalling pa hways [4,5]. Abno mal MT unc ion and exp ession ha e been implica ed in a ious human diseases, including cance [6]. The e a e a leas 10 iso o ms o MT in human body, which a e exp essed in a issue speci ic pa e n and may play dis inc oles in he a ious cell ypes. MT-I and MT-II iso o ms a e p esen in all cells h oughou he body, MT-III was i s isola ed as a g ow h inhibi ing ac o (GIF) om b ain neu ons, MT-IV is loca ed in s a i ied epi helium [6]. T ansc ip ion o MT-I and MT- -II can be induced by in lamma o y cy okines (IL-6, TNF-α, in e e ons), lipopolysaccha ids, glucoco icoids, ee adicals, an ioxidan s o Immunohis ochemical de ec ion o me allo hionein Gab iella Em i1*, Esz e Em i1, Li ia Beke2, Gábo Bo os1, Csaba Hegedűs1, Esz e Janka1, Emese Gellén1, Gábo Méhes2 and É a Remenyik1 1 Depa men o De ma ology, Facul y o Medicine, Uni e si y o Deb ecen, Nagye dei k . 98, H-4032 Deb ecen, Hunga y – Eu opean Union; E-Mails: [email p o ec ed]; bo [email p o ec ed]; hege- [email p o ec ed]; janka.esz e [email p o ec ed]; [email p o ec ed]; [email p o ec ed] deb.hu 2 Ins i u e o Pa hology, Facul y o Medicine, Uni e si y o Deb ecen, Nagye dei k . 98, H-4032 Deb ecen, Hunga y – Eu opean Union; E-Mails: [email p o ec ed]; [email p o ec ed]; * Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed]; Tel.: +36-52-255-602; Fax: +36-52-255-736. Recei ed:10.8.2015 / Accep ed:27.8.2015 / Published: 1.10.2015 Keywo ds: me allo hionein an ibodies; me allo hionein issue exp ession; immunohis ochemis y 1. In oduc ion Zinc (Zn (II)) is an essen ial mic oelemen , i has c i ical ole in no mal heal h and de elop- men [1]. Binding o Zn (II) o zinc coo dina ion mo i s in p o eins s abilizes he s uc u e o in luences he unc ion. The p e alence o genes encoding zinc p o eins is es ima ed o be o e 3% o he 32,000 iden i ied genes. O e 300 Zn (II)-dependen enzymes ha e been de ined and cha ac e ized [2]. I has been shown ha Zn (II) can egula e he DNA-binding ac i i y o zinc inge ansc ip ion ac o s [3]. The Zn (II)-me allo hionein (MT)/ hionein pai , which is an impo an componen o cellula Zn (II) homeos asis, is c i ical o seques e o elease Zn(II) depending on he local edox s a e, All human issues consis o a wide a ie y o cells ha ha e an impac on each o he . Human diseases ha a e caused by gene ic al e a ions and/o en i onmen al ac o s can be cha ac e ized by mo phological and unc ional changes in issues. Bo h aspec s a e impo an o be e unde s and he pa homechanism o a disease and o ind new he apeu ic a ge s. Immunohis ochemis y iden i ies he exp ession, in acellula localiza ion as well as issue dis ibu ion o a ious p o eins, while mo phology o issue can also be assessed p ecisely. I is used o ou ine diagnos ics as well as o esea ch. Howe e , an igen speci ic assay s anda diza ion and he use o app op ia e posi i e and nega i e issue con ols a e e y impo an o in e p e he indings in a way ha ensu es he biological ele ance. Abno mal me allo hionein unc ion and exp ession ha e been implica ed in a ious human diseases, including cance . Immunohis ochemis y p o ides an excellen oppo uni y o gain an insigh in o he ole o MT in he pa hogenesis o diseases. A icle 34 hea y me als. MT is a cy osolic p o ein in es- ing cells, bu i can be ansloca ed ansien ly o he cell nucleus du ing cell p oli e a ion and di e en ia ion [7]. Immunohis ochemis y (IHC) iden i ies he exp ession, in acellula localiza ion as well as issue dis ibu ion o a ious p o eins, whi- le mo phology o issue can also be assessed p ecisely [8]. IHC de ec ion o MT in issue samples is a e y impo an op ion o s udy i s ole in he pa hogenesis o diseases. Special IHC me hods such as mul iple immunolabeling using se ial sec ions o issue blocks o double s aining echnique p o ide an oppo uni y o s udy co ela ions be ween MT exp ession and impo an cell and issue unc ions [9]. Tissue mic oa ay allows simul aneous examina ion o la ge numbe o issues on a single mic oscope slide; he e o e i is e y sui able o e alua e he diagnos ic, p ognos ic o p edic i e ole o he MT exp ession [9]. 2.Immunohis ochemical de ec ion o me allo hionein The IHC echnique is a combina ion o immu- nologic and chemical eac ions isualized wi h a pho onic mic oscope [8]. I can be di ided in p e-analy ical, analy ical and pos -analy ical phases. I s a s wi h issue ixa ion, embed- ding, and issue sec ioning, ollowed by depa- a iniza ion, an igen e ie al, blocking o non- speci ic ac i i ies, incuba ion wi h he p ima y an ibody, and labeling o he an igen-an ibody eac ion, and ends wi h slide coun e s aining, co e slipping and e alua ion (Figu e 1.). Mos commonly, o malin- ixa ion and pa a in-em- bedding (FFPE) is used o i s abili y o p ese e issue inde ini ely o mo phologic examina ion [8,10]. Hea -induced epi ope e ie al was p o- ed o es o e he immuno eac i i y o issues ixed in o malin [8] Figu e 1: Ou line o s anda d immunohis oche- mical p o ocol [8,11,12]. 2.1 P e-analy ical phase o immunohis ochemis y The aim o ixa ion is o main ain mo phologi- cal ea u es and o p ese e issue sui able o a ange o s aining and IHC [11]. Adequa e issue ixa ion is e y impo an o s abilize p o eins and p e en issue decay. Tissue ixa ion may be accomplished by physical ( eeze) and/o chemical (coagula i e and c oss-linking) me- hods. Fo IHC pu poses, he ixa i e o choice is a 10% neu al-bu e ed o malin solu ion. The specimen should be placed in ixa i e im- media ely a e i is emo ed om he pa ien . The olume o o malin should be 10 o 20 imes he olume o specimen. Delayed, oo sho o oo long ixa ion can lead o alse IHC esul s [8]. Fo maldehyde pene a es he issue e y quickly, bu ixes i e y slowly; 16-24 h ixa ion ime is equi ed o a issue specimen o 1-4 mm in diame e . La ge specimens should be cu in o slices 4 o 5 mm hick o u he ixa ion [11]. Tissue p ocessing e e s o a se ies o s eps ha include emo al o he ex ac able wa e and lipids om he issue, usually in an au oma ed manne , hen in il a ion and embedding in a suppo ma ix (pa a in) so ha he issue can be s abilized and cu easily [11]. The specimens a e cu on a o a y mic o ome in o sec ions 3 o 5 μm hick. Sec ions ha will be used o IHC Jou nal o Me allomics and Nano echnologies 2015, 3, 33—42 35 should be moun ed on glass slides specially coa ed o cha ged o ensu e be e adhe ence [11]. Long- e m s o age o issue sec ions is no ecommended, because pho o-oxida ion o sec- ions esul s in loss o an igenici y [8]. De ec ion o ce ain an igens has he limi a ion o equi- ing esh issue. In his case he specimen is ob ained esh and kep mois un il i is o ien ed and embedded in op imal cu ing empe a u e (OCT) compound, hen he specimen is snap ozen [11]. The issue is hen sec ioned a 6 μm. Occasionally, special issue p ocessing (e.g., decalci ica ion, demelaniza ion) is equi ed p io o he immunos aining, bu epi opes can be des oyed by an agg essi e p ocedu e [8]. The s anda d o p o ocol o issue ixa ion and p ocessing mus be de eloped o each an igens and issue ype. I is wo h o highligh ha p e-analy ical ac o s in luence he success o a subsequen nucleic acid analysis as well [10]. 2.2 Analy ical phase o immunohis ochemis y Tissue sec ions moun ed on coa ed o cha ged glass slides a e depa a inized and ehyd a ed be o e an igen e ie al (AR). Incomple e depa- a iniza ion causes subop imal o incomple e s aining because o incomple e issue pene a i- on by he an ibody [12]. Hea - and/o p o ease- -induced AR is a p ocedu e which e e ses he s uc u al changes induced by he ixa ion and issue p ocessing [8]. AR echniques a e c i ical o an igen unmasking, op imal esul s equi e con ol o he pH and empe a u e o e ie al solu ions and con olled enzyma ic diges ion [12]. The mechanism o hea o es o e he im- muno eac i i y o o malin- ixed issues is no comple ely clea , he dissocia ion o i ele an p o eins om a ge pep ides, hyd olysis o me- hylene c oss-links, es o ing he na i e elec o- s a ic cha ges, mobiliza ion o ace pa a in e c. can be in ol ed [8]. Enzyma ic ea men is op imal only o ew an igens and can al e issue mo phology o des oy epi opes [8]. An igen epi opes consis o 5-6 amino acid esidues, and a e classi ied as linea o con o - ma ional [8]. An igens may consis o mul iple iden ical o dis inc epi opes. An igen-an ibody binding in ol es hyd ophobic, an de Waals, and elec os a ic in e ac ions. The bes diluen s bu e o p ima y an ibody is 0.05 o 0.1 M T is bu e (pH 6.0) [8]. Backg ound eac i i y due o ionic in e ac ions can be educed by inc easing he NaCl concen a ion in he bu e , bu i can educe he an igen-an ibody binding [8]. De e - gen s (e.g., Tween 20) a e also used o acili a e an igen-an ibody binding by solubilizing mem- b ane p o eins; hey a e usually inco po a ed in o dilu ion/ inse bu e s [8]. An ibody con- cen a es and p e-dilu ed p epa a ions mus be op imized o usage a he co ec dilu ion [12]. The a ini y o an an ibody o an an igen a ec s he sensi i i y and speci ici y o an immunolo- gical eac ion [8]. The o e all binding in ensi y be ween an ibodies and a mul i alen an igen is desc ibed by a idi y. Polyclonal an ibodies usually ha e highe a idi y, bu lowe speci ici y compa ed wi h monoclonal an ibodies [8]. The high speci ici y, howe e , does no elimina e he possibili y o c oss- eac i i y wi h o he an igens, because he a ge epi opes can be pa o mul iple p o eins and pep ides. The mos equen eason o he ailu e o IHC is he poo quali y o he p ima y an ibody used. Howe e , nonspeci ic backg ound s aining, less han op imal speci ic s aining, o no s aining equi e a ca e ul e alua ion o all componen s in ol ed in each s ep o he IHC echnique [13]. 2.2.1 De ec ion sys em P ima y (di ec de ec ion), seconda y o e i- a y (indi ec de ec ion) an ibodies labelled wi h enzymes, me als o luo escen compounds a e used o isualize he an igen-an ibody eac ion [8,12]. The de ec ion sys em mus be compa ible wi h he species es ed. The mos common label is he ho se adish pe oxidase, which p oduces a colou ed p ecipi a e a he si e o he an igen- -an ibody eac ion in he p esence o i s spe- ci ic subs a e and ch omogens. Endogenous pe oxidase ac i i y has o be blocked be o e incuba ion wi h p ima y an ibody. Ch omogen en apmen , p ecipi a ion and con aminan s may lead o alse-posi i e in e p e a ion o an IHC es [12]. Th ee-s ep echniques a e mo e sensi i e, e.g., he labelled s ep a idin-bio in (LSAB) me hod, which is widely used. In his case, a bio inyla ed seconda y an ibody binds Em i e al. 36 o he p ima y an ibody a ached o he issue an igen, and he bio inyla ed complex is de ec- ed by s ep a idin ha has been conjuga ed o he enzyme. Ne e heless, he endogenous bio in-associa ed backg ound s aining has led o inc easing use o labelled polyme -based de ec ion sys ems ha a e cha ac e ized by g ea e sensi i i y, speci ici y, and sui able o manual and au oma ed IHC pla o ms. Simul- aneous o sequen ial mul iple labelling using a ious de ec ion sys ems can be used o lo- calize di e en an igens in he same sec ions. Quan um do labelling is a p omising new ool o mul ispec al analysis [8]. F ozen sec ions can be mo e app op ia e o examine he exp ession o ce ain p o eins. In his case, immuno luo escence is used o de ec ion o an igen-an ibody eac ion. P i- ma y (o seconda y) an ibodies a e linked o a luo escen label such as luo escein iso hiocya- na e o allow isualiza ion using a luo escence mic oscope [12]. Compa ed o ozen sec ions, pa a in-embedded issues o e he ad an age o be e p ese a ion o cellula de ails and pe manency o he eac ion [11]. 2.3 Pos -analy ical phase o immunohis ochemis y Pos -analy ical phase o IHC includes assay s anda diza ion/ alida ion, con ol pe o - mance, and in e p e a ion o IHC esul s [8,14]. IHC assay s anda diza ion is impo an o ob- ain consis en and ep oducible esul s wi hin each labo a o y and compa able esul s among labo a o ies [8]. S anda diza ion is he p ocess o op imizing he es me hod ( eagen s and p o ocols). Guidelines o an igen-, issue- and species-speci ic s anda diza ion o IHC exami- na ions should be based on cu en ly a ailable published e idence and mode n unde s anding o quali y assu ance p inciples as applied o IHC in gene al [15]. Fu he mo e, diagnos- ic IHC labo a o ies mus mee he ISO 15189 s anda ds o s anda ds o ope a ion as de ined by he Clinical Labo a o y Imp o emen Amen- dmen s, espec i ely [16]. In non-acc edi ed labo a o ies, IHC es s should be alida ed by documen a ion o in e nal and ex e nal qua- li y assessmen s [17]. Use o quali y con ols is equi ed o echnical calib a ion and ana- ly ical alida ion [14]. Posi i e issue con ol is de ined as issue ha is known o con ain he an igen o in e es . Posi i e and nega i e issue con ols mus be ixed and s ained in he same way as he es ed specimen [8]. Posi i e issue con ol in he es ed specimen is designa ed as in e nal posi i e issue con ol, e.g., MT immu- no eac i i y in basal laye o no mal epide mis (Figu e 2.). Nega i e eagen con ols a e used o con i m he speci ici y o he es and o assess he deg ee o nonspeci ic backg ound s aining p esen by omi ing he p ima y an ibody [14]. Valida ion de ec s any c oss- eac i i y o he selec ed an ibodies wi h un ela ed an igens, and c oss- eac i i y among di e en issues and among di e en species, examines he a iables ha a ec he IHC eac ion, such as ixa ion ime and s o age o uns ained issue sec ions, and may include compa ison o esul s among di e en labo a o ies using simila echniques [8]. Whene e possible, alida ion compa es he sensi i i y o IHC de ec ion o he gold s anda d me hod o de ec ion o he Ag in ques ion. Acco ding o he Clinical and Labo a o y S an- da ds Ins i u e sugges ions, he IHC epo should include he cellula loca ion and issue dis ibu ion o he es ed an igen, semi-quan i- a i e e alua ion o he immuno eac i i y, along wi h an in e p e a ion o he es esul s [8,13- 15]. The epo should con ain demog aphic in o ma ion, he issue ha was es ed, disease cha ac e is ics, and he an ibody used. IHC sco ing schemes a e based on a subjec i e as- sessmen o he labelling in ensi y and pe cen posi i e cells by a pa hologis . Recen ly, i has been demons a ed ha so wa e algo i hms a e able o p ope ly indica e he disease- ele an egions in digi ized issue images and quan i y he a ea and op ical densi y o posi i e s aining [18,19]. Ad an ages o an au oma ed digi al IHC image analysis a e ha i is unbiased, p ecise in anges o s aining ha appea weak o he eye, and p oduces con inuous da a [18]. 2.4 An ibodies o me allo hionein immunode ec ion In mammals, ou andemly clus e ed MT genes a e known [20]. In humans, MT-I has Jou nal o Me allomics and Nano echnologies 2015, 3, 33—42 37 unde gone duplica ion e en s ha ha e esul ed in 13 duplica e iso o ms, i e o which ha e been p edic ed o be no ac i e o ms [20]. All genes encode o conse ed pep ide chains ha e ain 20 in a ian me al-binding cys eines. A o al o 9 and 11 cys eines a e equi ed o o m p o- ein domains ha bind h ee and ou di alen me al ions [21]. MT-I and MT-II a e 61- esidue p o eins, MT-III holds an ex a esidue in he β-domain and a six- esidue long inse ion in he α-domain, whe eas MT-IV sha es an addi ional esidue in he β-domain [20]. In humans, he ansc ip ion o MT-I and MT-II iso o ms is induced by a ious s imuli [21]. The exp ession o MT-II, MT-IE and MT-IX can be de ec ed in mos ypes o issue, whe eas he exp ession o o he MT-I iso o ms seems o be es ic ed o some issues [20]. MT-III is cons i u i e issue- -speci ic iso o m, i is exp essed in human b ain and in some o he issues [20]. MT-IV mRNA exp ession has been epo ed in s a i ied squa- mous epi helium in mouse, no da a exis on he exp ession o MT-IV in humans [20]. In o de o in es iga e p o ein exp ession, he mos widely used p ima y an ibody o de ec MT-I/II p o eins in FFPE issues o a numbe o di e en species is a monoclonal mouse an- ibody (Clone E9, Dako) eac ing wi h a single and highly conse ed epi ope o med by he las 5-7 esidues o he N- e minus o he β-domain o MT-I and MT-II [22-25]. The immunogen was ho se sel -polyme ized MT-I and MT-II. In p e- ious s udies a speci ic polyclonal an ibody ha was gene a ed agains a li e MT in abbi s was used e icien ly o de ec MT exp ession also in humans [7,26,27]. Fu he mo e, due o he di e ence in he amino acid sequence, speci ic monoclonal and polyclonal an ibodies agains MT-III could be gene a ed and used success ully in IHC es s [22,28-30]. Fu he p og ess in he de elopmen o p ima y an ibo- dies agains MT iso o ms would be impo an o u u e in es iga ions. Ne e heless, IHC s udies o da e ha e g ea ly con ibu ed o he cu en knowledge on he unc ions o MT in he human body. 2.5 Tissue speci ic exp ession o me allo hionein p o eins MT-I and MT-II can be exp essed in all cells h oughou he body, howe e , IHC examina i- ons we e able o e eal ha no all cells exp ess MT o he same ex en in heal hy issues [27]. In addi ion, se e al ac o s, e.g., p oli e a ion s a us, age, can in luence he exp ession o MT. Fo example, in no mal human skin, s ong MT-I/II immunos aining can be de ec ed in ba- sal ke a inocy es o epide mis and hai ollicle ou e oo shea h, hai ma ix cells and he sec e o y coil, bu no in he exoc ine po ion o ecc ine glands [31]. In hype plas ic epide - mis he inc eased MT exp ession ep esen s an inc ease in he ge mina i e pool size [32]. Acco dingly, he p o ein le els o MT-I and MT-II ha e been shown o dec ease signi ican ly wi h inc easing age simul aneously wi h he dec ease o ke a inocy e p oli e a ion [33]. Na ey e al. in es iga ed he dis ibu ion o MT du ing human de elopmen [34]. They ound ha MT le els a e highe in he e al li e han in he adul li e ; in addi ion, MT is localized in he nucleus and he cy oplasm o human e al and neona al hepa ocy es, whe eas MT is mainly exp essed in he cy oplasm o adul li e cells. In he e al and neona al human kidney, MT is localized p ima ily in he nucleus and he cy oplasm o he p oximal ubula epi helial cells, whe eas in he adul kidney, in aluminal MT localiza ion was also obse ed [34]. MT-III exp ession has been in es iga ed by IHC in a s [28]. Some as ocy es in he deep laye s o co ex, ependymal cells, some glo- me ula and ubula cells in he kidney, some glandula epi helial cells in he do sola e al lobe o p os a e, some Se oli cells and Leydig cells in he es is, and as e bud cells in he ongue showed MT-III immunos aining [28]. 2.5.1 Me allo hionein exp ession in esponse o exogenous agen s I is hough ha MTs exe cy op o ec i e e ec s agains hea y me al oxici y and oxi- da i e s ess [27]. IHC is sui able me hod o in es iga e he exp ession o MT in a ious issues in esponse o an exogenous agen . An example is he ele a ed nuclea and cy oplasmic Em i e al. 38 MT exp ession o pe ipo al hepa ocy es, p oxi- mal ubula epi helial cells, in es inal columna epi helial cells and Pane h cells in a s du ing die a y coppe o zinc o e loads, and upon exposu e o cadmium [25,35,36]. Fu he mo e, he hepa ic MT le els we e inc eased in bo h he nucleus and cy oplasm o hepa ocy es in mice upon hioace amide exposu e [37], and in a s upon exposu e o 2,3,7,8- e achlo odi- benzo-p-dioxin [38]. Inc eased MT-I and MT-II exp ession in he enal p oximal ubula cells o a s upon adminis a ion o doxo ubicin migh indica e a p o ec i e mechanism agains he p o-apop o ic e ec o his cy os a ic agen [39]. The MT le els ha e been ound o be ele a ed in he epide mis a e acu e ul a iole adia ion exposu e [40]. 2.5.2 Me allo hionein exp ession in in lamma o y condi ions MT knockou mice appea ela i ely heal hy, bu hey show an impai ed esponse o s ess [5]. MT exp ession is induced by a a ie y o p o- and an i-in lamma o y media o s inclu- ding glucoco icoids, eac i e oxygen species, an ioxidan s, endo oxin, acu e phase cy okines, u he mo e, MT ha e a wide ange o unc ions in cellula homeos asis and immuni y [41]. Al e ed exp ession o MT has been shown in many in lamma o y condi ions. Fo example, an IHC s udy on enal biopsies om pa ien s wi h lupus neph i is showed he deple ion o MT-I and MT-II p o ein exp ession in he p oximal ubula epi helial cells compa ed wi h heal hy kidney specimens [42]. Mo eo e , a ubula MT sco e below he median alue o he coho p e- dic ed a poo enal ou come. Tissue in il a ing MT-posi i e cells could be shown in colonic biopsies om pa ien s wi h in lamma o y bo- wel disease and acu e in ec ious coli is [43]. In ano he s udy, he mucosal MT concen a ion in colonic issue samples om pa ien s wi h in lamma o y bowel disease has been ound o be lowe compa ed wi h no mal mucosa issue samples sugges ing an an ioxidan imbalance in he in es inal mucosa o hese pa ien s [44,45]. In a dex an sul a e sodium-induced in es inal in lamma ion mouse model MT-posi i e cells we e de ec ed in he lamina p op ia and sub- mucosal laye and we e mainly co-localized in mac ophages [46]. Ne e heless, he inc ease o colonic myelope oxidase ac i i y le els and p o-in lamma o y cy okine p oduc ion induced by dex an sul a e sodium was signi ican ly highe in he MT-I/II knockou animals com- pa ed wi h he wild- ype mice sugges ing ha MT plays a p o ec i e ole agains coli is [46]. MT exp ession was analyzed in he cen al ne ous sys em o mice wi h expe imen al au o- immune encephalomyeli is [47]. MT-I/II seemed o be p oduced mainly by eac i e as ocy es and ac i a ed mac ophages. I was assumed ha he ele a ion o MT exp ession le el in issue may ac on he in lamma o y mic oen i on- men supp essing p o-in lamma o y cy okine p oduc ion o mac ophages, dec easing apop o- ic cell dea h in neu ons and oligodend ocy es, and enhancing issue epai [47]. 2.5.3 Me allo hionein exp ession in cance MT exp ession in issues has been s udied mos in ensi ely in human cance s [48]. The esul s sugges ha change in MT le els migh play a ole in he con e sion o a po en ially malignan lesion o a malignan ca cinoma. Fo example, signi ican ly highe MT-I/II and MT-III exp ession was no ed in ac inic ke a osis and cu aneous squamous cell cance , as compa- ed wi h no mal skin epide mis, whe eas e y low le els o MT-III exp ession we e ound in basal cell cance [22,29]. A signi ican ly highe MT-I/II exp ession was also obse ed in o al squamous cell ca cinoma issues compa ing wi h no mal and o al leukoplakia epi helial issues [49]. High MT exp ession was de ec ed in panc eas adenoca cinoma issues compa ed wi h panc ea ic se ous cys adenoma o heal- hy panc ea ic issue samples [50]. Analysis o MT-I/II exp ession in p os a e epi helial cells showed highe MT exp ession in issues de i ed om benign p os a ic hype plasia han in hose de i ed om p os a e cance [51]. Fu he - mo e, IHC analysis o specimens om no mal colo ec al mucosa, adenomas, ca cinomas and lymph node me as ases e ealed he down- e- gula ion o MT-I/II exp ession in associa ion wi h colo ec al cance p og ession [52]. Janssen e al. con i med ha he MT concen a ions Jou nal o Me allomics and Nano echnologies 2015, 3, 33—42 39 o colo ec al adenomas, ca cinomas and li e me as ases a e lowe han ha o co esponding no mal mucosa, howe e , hey ha e ound ha a ela i ely high MT con en migh be associa ed wi h he agg essi eness o colo ec al cance s [53]. Me a-analysis o IHC s udies led o he conclusion ha signi ican ly inc eased MT-I/ II exp ession can be de ec ed in head and neck, and o a ian cance s, espec i ely, compa ed wi h heal hy issues, bu signi ican ly dec eased MT exp ession can be obse ed in li e umou s compa ed wi h no mal li e issue [54]. I seems ha he exp ession o MT is no uni e sal o all human umou s, bu may depend on he di e en ia ion s a us and p oli e a i e index o umou s, along wi h o he issue ac o s and gene mu a ions [48]. The a ious umou s may also di e in he in acellula localiza ion o MT. Fo example, we obse ed nuclea MT-I/II s ai- ning in benign melanocy ic ne i and in ensi e cy oplasmic and nuclea s aining in malignan melanomas (Figu e 2.) [9]. Cy oplasmic and nuclea MT-I/II exp ession was signi ican ly highe in endome ial cance cells compa ed wi h cells in benign hype plasia o endome- ium, howe e , he nuclea MT exp ession co ela ed be e wi h his ologic g ade [55]. Changes in MT exp ession (up- o down- egu- la ion) may be associa ed wi h a mo e agg essi e pheno ype and he apeu ic esis ance, ul ima e- ly esul ing in a wo se p ognosis [6,21]. Weinlich e al. ha e ound ha high MT le els in umou cells a e associa ed wi h educed su i al in pa ien s wi h malignan melanoma [56]. We con i med ha high exp ession o MT-I/II in melanoma cells is signi ican ly mo e equen in p ima y cu aneous malignan melanoma wi h haema ogenous me as ases [9]. In bladde cance pa ien s a high MT exp ession in umou issues was linked o sho e umou -speci ic su i al and inc eased ecu ence a es [57]. IHC analysis o specimens om enal cell ca - cinoma e ealed ha MT immunos aining is associa ed wi h signi ican ly wo se p ognosis [58]. The absence o MT exp ession in o a i- an cance samples co ela ed wi h imp o ed p og ession- ee su i al in pa ien s ea ed wi h adju an pla inum-based chemo he apy [59]. Ne e heless, he ole o MT in me as asis o ma ion emains o be con i med, and ex- pe imen al e idence o i s oncogenic ole is s ill lacking. Figu e 2: Exp ession o MT-I/II in p ima y cu aneous malignan melanoma cells (a ows). The ke a inocy es (as e isks) o epide mal basal laye se ed as in e nal posi i e issue con ols. The IHC de ec ion was based on an immunope oxidase eac ion using VIP ch omogenic subs a e. The slides we e coun e s ained wi h me hyl-g een. O iginal magni ica ion is x20. 3. Conclusions Zn (II) is essen ial in ine- uned o ches a ion o basic cell unc ions such as p oli e a ion, gene exp ession and s ess esponse. Besides me al de oxi ica ion, MT can elease/bind Zn (II) wi h high a ini y and egula es he a ailabili y o Zn (II) in a ious cell compa men s he eby in luencing he unc ion o many ansc ip ion ac o s and enzymes [5]. I migh explain why we can de ec changes in he exp ession o MT in cance , and calls o u he in es iga ions in his ield. Mo eo e , he a ailabili y o zinc om MT is con olled by he local edox s a- us [4]. Al e a ions in eac i e oxygen species homeos asis a e hough o be in ol ed in he pa hogenesis o a ious diseases, including cance s [6]. IHC is sui able o demons a e MT exp ession in i s mo phological and unc ional Em i e al. 40 con ex ; he e o e, i is an impo an elemen o he in es iga ions. Assay s anda diza ion and he use o app op ia e posi i e and nega i e issue con ols a e e y impo an o in e p e he indings co ec ly. Acknowledgmen s This wo k was suppo ed by he Eu opean Social Fund TÁMOP-4.2.2.A-11/1/KONV-2012- 0031, he Hunga ian Scien i ic Resea ch Fund OTKA K68401 and NK101680, and he Me a- llomic Scien i ic Ne wo k V4MSNe (p ojec 11440027). Con lic s o In e es The au ho s decla e no con lic o in e es . The au ho s decla e hey ha e no po en ial con lic s o in e es s conce ning d ugs, p o- duc s, se ices o ano he esea ch ou pu s in his s udy. The Edi o ial Boa d decla es ha he manusc ip me he ICMJE „uni o m egui e- men s“ o biomedical pape s. Re e ences 1. Schwa z, J.R.; Ma sh, R.G.; D aelos, Z.D. Zinc and skin heal h: O e iew o physiology and pha macology. 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