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.
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