O osz e al. Jou nal o Biomedical Science 2010, 17:47
h p://www.jbiomedsci.com/con en /17/1/47
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RESEARCH
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Resea ch
In ol emen o p63 in he
he pes simplex
i us
-1-induced demise o co neal cells
László O osz
1
, É a Gallyas
2
, Lajos Kemény
3,4
, Y e e Mándi
1
, And ea Facskó
5
and Klá a Megye i*
1
Abs ac
Backg ound: The ansc ip ion ac o p63 plays a pi o al ole in he de elopmen and main enance o epi helial
issues, including he ocula su ace. In an e o o gain insigh in o he pa hogenesis o ke a i is caused by HSV-1, we
de e mined he exp ession pa e ns o he p63 and Bax p o eins in he S aa ens Se umins i u e Rabbi Co nea cell line
(SIRC).
Me hods: SIRC cells we e in ec ed wi h HSV-1 a a ious mul iplici ies and main ained o di e en pe iods o ime.
Vi us eplica ion was measu ed by indi ec immuno luo escence assay and Wes e n blo analysis. Cell iabili y was
de e mined by MTT assay. The apop o ic esponse o he in ec ed cells was quan i ied by ELISA de ec ing he
en ichmen o nucleosomes in he cy oplasm. Wes e n blo analysis was used o de e mine he le els o p63 and Bax
p o eins.
Resul s: Indi ec immuno luo escence assays and Wes e n blo analyses demons a ed he p esence o HSV-1
glycop o ein D (gD) in he in ec ed SIRC cell line, and he pa e n o gD exp ession was consis en wi h e icien i al
eplica ion. The esul s o MTT and ELISA assays showed ha HSV-1 elici ed a s ong cy opa hic e ec , and apop osis
played an impo an ole in he demise o he in ec ed cells. Mock-in ec ed SIRC cells displayed he cons i u i e
exp ession o ΔNp63α. The exp essions o he Bax-β and TAp63γ iso o ms we e conside ably inc eased, whe eas he
le el o ΔNp63α was dec eased in he HSV-1-in ec ed SIRC cells. Expe imen s in ol ing he use o acyclo i showed
ha i al DNA eplica ion was necessa y o he accumula ion o TAp63γ.
Conclusion: These da a sugges ha a di ec , i us-media ed cy opa hic e ec may play an impo an ole in he
pa hogenic mechanism o he pe ic ke a i is. By dis u bing he delica e balance be ween he p o-su i al ΔN and he
p o-apop o ic TA iso o ms, HSV-1 may cause p o ound al e a ions in he iabili y o he ocula cells and in he issue
homeos asis o he ocula su ace.
Backg ound
The p53 amily membe p63 has been shown o play a
pi o al ole in he homeos a ic enewal o epi helial is-
sues [1-3]. The e a e six p63 p o ein iso o ms, which can
be exp essed om wo di e en p omo e s, one immedi-
a ely p eceding he i s exon and he second one lying in
he hi d in on (Fig. 1) [1-8]. T ansc ip ion om he i s
and second p omo e s gi es ise o TA- o ΔN-amino-
e mini o p63, espec i ely (Fig. 1) [1-8]. The TA iso-
o ms possess an N- e minal acidic ansac i a ion
domain, while he ΔNp63 p o eins lack his domain (Fig.
1) [1-8]. A g ea body o expe imen al e idence indica es
ha he TAp63 iso o ms can induce cell dea h h ough a
canonical p53- esponsi e DNA binding si e [1-12]. In
con as , he ΔNp63 p o eins can ac in a dominan nega-
i e manne owa d p53-media ed ansc ip ional ac i a-
ion [1-12]. Bo h TA and ΔN ansc ip s can unde go
al e na i e splicing, leading o he o ma ion o h ee C-
e minal a ian s, deno ed α, β and γ, which u he
inc ease he di e si y o he p63 p o eins (Fig. 1) [1-8].
Se e al in e es ing s udies ha e clea ly demons a ed ha
he ΔNp63α iso o m plays an impo an ole in he main-
enance o he conjunc i al and co neal s em cells, while
ΔNp63β and ΔNp63γ con ibu e o he egula ion o cell
di e en ia ion and egene a ion in he conjunc i a, lim-
bus and co nea [13-19]. Al hough he impo ance o p63
in he homeos asis o he ocula su ace is widely
accep ed, he e ec s o in ec ious agen s on he exp es-
* Co espondence: megye i@comse .szo e.u-szeged.hu
1 Depa men o Medical Mic obiology and Immunobiology, Uni e si y o
Szeged, Dóm é 10, H-6720 Szeged, Hunga y
Full lis o au ho in o ma ion is a ailable a he end o he a icle
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sion o his ansc ip ion ac o amily ha e no ye been
in es iga ed in epi helial cells o he eye.
He pe ic ke a i is is a ision- h ea ening i al disease o
he eye ha is he majo in ec ious cause o blindness in
he de eloped coun ies [20-22]. The causa i e agen ,
He pes simplex i us 1 (HSV-1) is a membe o he He -
pes i idae amily comp ising la ge, en eloped DNA
i uses [23]. P ima y he pe ic ke a i is can de elop
di ec ly ia ' on -doo ' ou e in ec ion by d ople sp ead,
o ia a 'back-doo ' ou e, which in ol es he indi ec
sp ead o HSV-1 o he co nea om a non-ocula si e
[20]. HSV-1 in ec ion may a ec all h ee co neal laye s,
leading o epi helial, s omal and endo helial ke a i is,
espec i ely. Epi helial ke a i is can be cha ac e ized by
he appea ance o b anching dend i i o m, o enla ged
geog aphic ulce s [21]. S omal ke a i is and endo heli is
can esul in s omal sca ing, hinning, neo ascula iza-
ion, se e e i idocycli is and an ele a ed in aocula p es-
su e [20]. Mos cases o co neal ulce a ion will e en ually
esol e, hough ecu en in ec ions impai he co neal
unc ion and lead o a ision impai men ha may e en
necessi a e pene a ing ke a oplas y. P e ious s udies
ha e e ealed ha he mechanism o he pe ic ke a i is
in ol es bo h immune- and i us-media ed cy opa ho-
genic p ocesses [24-28]. Whe eas he immune p ocesses
in ol ed in he pa hogenesis o he pe ic ocula su ace
diseases ha e been in es iga ed ex ensi ely, he molecula
e en s implica ed in he di ec cy opa hic ac ion o HSV-
1 emain la gely unknown.
In he p esen s udy, we examined he e ec s o HSV-1
on he exp ession o p63 and he Bcl-2 amily membe
Bax in an e o o gain a be e unde s anding o he ocu-
la cy opa hogenici y elici ed by his i us.
Me hods
Cell cul u e and HSV-1 g ow h
The S aa ens Se umins i u e Rabbi Co nea (SIRC) cell
line, was g own in Dulbecco's modi ied Eagle's minimal
essen ial medium (Sigma Chemical Co., S . Louis, MO,
USA) supplemen ed wi h 10% e al cal se um (Gibco/
BRL, G and Island, NY, USA) a 37°C in a 5% CO2 a mo-
sphe e.
The KOS s ain o HSV-1 was p opaga ed a a mul i-
plici y o in ec ion (MOI) o 0.001 plaque- o ming uni
(PFU) pe cell in Ve o cell cul u es o 3 days a 37°C. The
cul u e luid o HSV-1-in ec ed Ve o cells was ha es ed,
quan i ied by plaque assay, s o ed a -70°C, and used as
he in ec ing s ock o he i us.
Fo expe imen s, SIRC cell cul u es we e inocula ed
wi h HSV-1 a di e en MOIs. 9-[(2-Hyd oxy-
e hoxy)me hyl]guanine [Acyclo i (ACG); (Sigma)] was
used a a ious concen a ions when indica ed. E e y
expe imen was epea ed a leas h ee imes.
Indi ec Immuno luo escence assay
Cy ospin cell p epa a ions we e ixed in me hanol-ace-
one (1:1) o 15 minu es (min) a -20°C. Slides we e incu-
ba ed wi h a 1:200 dilu ion o polyclonal abbi an i-HSV
glycop o ein D (gD) immunoglobulin (Sigma) o 1 h a
37°C. A e washing wi h phospha e-bu e ed saline
(PBS), he samples we e eac ed wi h luo escein iso hio-
cyana e-conjuga ed an i abbi an ibody (1:160) (Sigma)
and incuba ed o 1 h a 37°C. A e washing wi h PBS,
he slides we e isualized by con ocal mic oscopy. The
a io o posi i e o nega i e cells was de e mined a e
coun ing 1,000 cells in andom ields.
Quan i ica ion o cell iabili y by MTT assay
The iabili y o HSV-1-in ec ed cells was measu ed wi h
he colo ime ic MTT [3-(4,5-dime hyl hiazol-2-yl)2,5-
diphenyl e azolium b omide] assay Tox-1 ki (Sigma). In
his assay, SIRC cells we e seeded in 96-well pla es a a
densi y o 1 × 104/well. The cul u es we e in ec ed wi h
HSV-1 a di e en MOIs. A 48 h pos in ec ion a 37°C,
10 μl MTT eagen (5 mg/ml) was added o each well.
A e 2 h incuba ion, MTT sol en con aining 0.1 M HCl
and isop opanol was added o 15 h. Abso bance was
measu ed a 545 and 630 nm. The a io o li ing cells was
calcula ed ia he ollowing o mula: pe cen age iabili y
= [(abso bance o in ec ed cells - blank)/(abso bance o
Figu e 1 (A) Gene a chi ec u e o human p63. The al e na i e p o-
mo e s and spicing e en s used o gene a e he a ious p63 iso o ms
a e indica ed. (B) Domain s uc u e o he a ious p63 p o eins. The
ansc ip ion ac i a ion domain (TAD), DNA binding domain (DBD), oli-
gome isa ion domain (OD), s e ile α mo i (SAM) and he ansinhibi o
domain (TID) a e depic ed. The molecula size o each iso o m is indi-
ca ed on he igh . (No d awn o scale; adap ed om [2-7]). aa, amino
acid
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co esponding mock-in ec ed con ol cells - blank)] ×
100.
Quan i ica ion o apop osis by enzyme-linked
immunoso ben assay (ELISA)
The cells we e washed in phospha e bu e ed saline (PBS)
and he cell pelle was p ocessed in a cell dea h de ec ion
ELISA ki (Roche Diagnos ics GmbH, Penzbe g, Ge -
many) based on he measu emen o his ones complexed
wi h mono- and oligonucleosome agmen s o med du -
ing cell dea h. Fo his assay, he cells we e incuba ed in
lysis bu e o 30 minu es (min) and cen i uged a
12,000 pm o 10 min. The supe na an s we e ans-
e ed in o a s ep a idin-coa ed mic opla e and incu-
ba ed wi h bio in-conjuga ed an i-his one and
pe oxidase-conjuga ed an i-DNA monoclonal an ibodies
o 2 h. A e washing, subs a e solu ion 2,2'-azino-bis(3-
e hylbenz hiazoline-6-sulphonic acid) (ABTS) was added
o each well o 15 min. Abso bance was measu ed a 405
and 490 nm. The speci ic en ichmen o mono- and oligo-
nucleosomes was calcula ed as en ichmen ac o (EF) =
abso bance o HSV-1-in ec ed cells/abso bance o co e-
sponding non-in ec ed con ol cells.
Wes e n blo assays
Cells (1 × 107) we e homogenized in ice-cold lysis bu e
con aining 150 mM NaCl, 10 mM T is HCl, pH 7.6, 5
mM EDTA, 1% ( / ) Nonide P-40, 0.1% SDS, 1% sodium
deoxychola e and p o ease inhibi o cock ail (Sigma), and
he mix u e was hen cen i uged a 10,000 g o 10 min o
emo e cell deb is. P o ein concen a ions o cell lysa es
we e de e mined by using he Bio-Rad p o ein assay (Bio-
Rad, He cules, CA, USA). Supe na an s we e mixed wi h
Laemmli's sample bu e and boiled o 3 min. Aliquo s o
he supe na an s, con aining 50 μg o o al p o ein o
de ec p63, HSV D glycop o ein (gD) and Bax, we e
esol ed by SDS-PAGE and elec o ans e ed on o ni o-
cellulose il e s (Ame sham, Buckinghamshi e, UK). P e-
blocked blo s we e eac ed wi h speci ic an ibodies o
HSV gD (Sigma), p63 de ec ing all o he a ious p63 iso-
o ms (clone 4A4) (San a C uz Bio echnology Inc., Cam-
b idge, MA, USA), p40 de ec ing he ΔNp63 iso o ms
(Me ck KGaA, Da ms ad , Ge many) and Bax (Pha Min-
gen, SanDiego, CA, USA) o 4 h in PBS con aining 0.05%
( / ) Tween 20, 1% (w/ ) d ied non- a milk (Di co Labo-
a o ies, De oi , MI, USA) and 1% (w/ ) BSA [ ac ion V;
(Sigma)]. Blo s we e hen incuba ed o 2 h wi h species-
speci ic seconda y an ibodies coupled o pe oxidase [pe -
oxidase-conjuga ed an i-mouse an ibody (DakoCy oma-
ion, Ca pin e ia, CA, USA), o pe oxidase-conjuga ed
an i- abbi an ibody (DakoCy oma ion)]. Fil e s we e
washed i e imes in PBS-Tween o 5 min a e each s ep
and we e de eloped by using a chemiluminescence de ec-
ion sys em (Ame sham). The au o adiog aphs we e
scanned wi h a GS-800 densi ome e (Bio-Rad), and he
ela i e band in ensi ies we e quan i ied by use o he
ImageQuan so wa e (Ame sham).
S a is ical analysis
All alues a e exp essed as means ± s anda d de ia ion
(SD). The one-way ANOVA es wi h he Bon e oni
pos - es was used o pai wise mul iple compa isons,
and P alues < 0.05 we e conside ed s a is ically signi i-
can .
Resul s
HSV-1-in ec ed SIRC cells exhibi gD exp ession and
inc eased apop o ic a es
The SIRC cell line was in ec ed wi h he KOS s ain o
HSV-1 a a ious mul iplici ies and main ained o di e -
en pe iods o ime.
Indi ec immuno luo escence assays o e alua e HSV-1
eplica ion e ealed posi i e s aining o gD a 48 h
pos in ec ion (hpi) in ≥ 99% o SIRC cells in ec ed a an
MOI o 1 (Fig. 2).
MTT assays o e alua e he cy opa hogenici y o HSV-1
e ealed dec eased iabili y a 48 hpi in 23 and 36% o
SIRC cells in ec ed a MOIs o 1 and 10, espec i ely (Fig.
3).
ELISA o e alua e he ex en o apop osis e ealed
inc eased apop o ic a es in HSV-1-in ec ed SIRC cells a
48 hpi; he EFs measu ed a MOIs o 0.1, 1 and 10 we e
1.42, 4.35 and 5.8, espec i ely (Fig. 3).
Toge he , hese da a demons a e he exp ession o
HSV-1 gD p o ein ha is consis en wi h e icien i al
eplica ion. Mo eo e , hese esul s e eal ha HSV-1
elici s a s ong cy opa hic e ec in he SIRC cell line, and
apop osis plays an impo an ole in he demise o he
in ec ed cells.
Figu e 2 Replica ion o HSV-1 in he SIRC cell line. SIRC cells we e
in ec ed wi h he KOS s ain o HSV-1 a an MOI o 1 o 48 h (B). Mock-
in ec ed SIRC cells cul u ed in pa allel we e le un ea ed (A). HSV-1
eplica ion was examined by con ocal mic oscopy a e s aining wi h
an HSV gD p o ein-speci ic abbi polyclonal an ibody p epa a ion and
FITC-conjuga ed an i- abbi immunoglobulin. Resul s a e ep esen a-
i e o h ee independen expe imen s.
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HSV-1 al e s he le els o Bax and p63 p o eins
To de e mine whe he HSV-1 can al e he exp essions o
Bax and p63, he s eady-s a e le els o hese p o eins we e
de e mined by Wes e n blo analysis.
Fi s , he kine ics o HSV-1 gD exp ession was in es i-
ga ed. The p esence o gD was obse ed in he SIRC cell
cul u es in ec ed wi h HSV-1 a an MOI o 10 a 12 hpi
(Fig. 4; lane 20). The gD p o ein accumula ed in he cul-
u es in ec ed wi h HSV-1 a MOIs o 0.1, 1 and 10 a 24
hpi (Fig. 4; lanes 23-25). High-le el exp ession o he gD
p o ein was also e ealed in e e y cul u e in ec ed wi h
HSV-1 by 48 hpi (Fig. 4; lanes 27-30).
The analysis e ealed he p esence o a Bax iso o m
co esponding o Bax-β in HSV-1-in ec ed SIRC cul u es
a 12 hpi ( he ela i e quan i y o Bax-β in cells in ec ed a
an MOI o 10 was 1.67) (Fig. 4; lane 20). A he 24-h ime
poin , he exp ession o he Bax-β p o ein in he HSV-1-
in ec ed SIRC cul u es was up egula ed ( he ela i e
quan i ies o Bax-β in cells in ec ed a MOIs o 1 and 10
we e 6.42 and 8.31, espec i ely) (Fig. 4; lanes 24 and 25).
A he 48-h ime poin , he HSV-1-in ec ed SIRC cul u es
displayed ele a ed le els o Bax-β ( he ela i e quan i ies
o Bax-β in cells in ec ed a MOIs o 0.01, 0.1, 1 and 10
we e 9.27, 9.93, 7.57 and 6.62, espec i ely) (Fig. 4; lanes
27-30).
The exp ession pa e n o p63 was de e mined by using
an an ibody p epa a ion which ecognizes all o he a i-
ous p63 iso o ms. The analysis e ealed he cons i u i e
exp ession o a p63 p o ein mig a ing nea 68 kDa in he
mock-in ec ed SIRC cells (lanes 1, 6, 11, 16, 21 and 26 in
Fig. 4). P e iously published da a demons a ed ha he
68 kDa p o ein possibly co esponds o ΔNp63α [4]. A
12 hpi, he exp ession o ΔNp63α in he HSV-1-in ec ed
SIRC cul u es was down egula ed ( he ela i e quan i y o
ΔNp63α in cells in ec ed a an MOI o 10 was 0.87) (Fig.
4; lane 20). A he 24-h ime poin , HSV-1 igge ed an
imp essi e educ ion in he le el o ΔNp63α in he SIRC
cells ( he ela i e quan i ies in cells in ec ed a MOIs o
0.01, 0.1, 1 and 10 we e 0.89, 0.43 and 0.41, espec i ely)
(Fig. 4; lanes 23-25). A he 48-h ime poin , he HSV-1-
in ec ed SIRC cul u es exhibi ed dec eased le els o
ΔNp63α ( he ela i e quan i ies in cells in ec ed a MOIs
o 0.01, 0.1, 1 and 10 we e 0.36, 0.22, 0.19 and 0.17,
espec i ely) (Fig. 4; lanes 27-30).
The expe imen s also e ealed he p esence o a 51-62
kDa p o ein in HSV-1-in ec ed SIRC cul u es. P e iously
published da a demons a ed ha he 51-62 kDa p o ein
possibly co esponds o TAp63γ [4]. A 12 hpi, HSV-1-
in ec ed SIRC cells exhibi ed inc eased le els o TAp63γ
( he ela i e quan i y o TAp63γ in cells in ec ed a an
MOI o 10 was 48.6) (Fig. 4; lane 20). A he 24-h ime
poin , he exp ession o TAp63γ in he HSV-1-in ec ed
SIRC cul u es was highly up egula ed ( he ela i e quan-
i ies in cells in ec ed a MOIs o 0.1, 1 and 10 we e 4.5,
78.1 and 82.4) (Fig. 4; lanes 23-25). A 48-h pos in ec ion,
he HSV-1-in ec ed SIRC cul u es displayed ele a ed le -
els o TAp63γ ( he ela i e quan i ies in cells in ec ed a
MOIs o 0.01, 0.1, 1 and 10 we e 81.8, 77.5, 75.6 and 63.4,
espec i ely) (Fig. 4; lanes 27-30).
To iden i y he p63 iso o ms, he s eady-s a e le els o
hese p o eins we e de e mined by Wes e n blo analysis,
using a polyclonal an ise um which eac s only wi h he
Figu e 3 HSV-1 induces cell dea h in he SIRC cell line. SIRC cells
we e in ec ed wi h HSV-1 a di e en MOIs o 48 h. Mock-in ec ed cells
cul u ed in pa allel we e le un ea ed. The cell iabili y was measu ed
by using he MTT assay (A). Apop osis was de ec ed by measu ing he
speci ic en ichmen o mono- and oligonucleosomes in he cy oplasm
by ELISA (B). The en ichmen ac o was calcula ed as he abso bance
o HSV-1-in ec ed cells/abso bance o co esponding non-in ec ed
con ol cells. Da a a e mean (± SD) alues om ou independen ex-
pe imen s. P alues we e calcula ed by he ANOVA es wi h he Bon-
e oni pos - es . aP < 0.001 s. mock; bP < 0.001 s. 0.1 MOI; cP < 0.001
s. 1 MOI; ns = nonsigni ican s. mock.
Figu e 4 HSV-1 in ec ion al e s he le els o p63 and Bax-β in he
SIRC cell line. To al p o ein was isola ed om mock-in ec ed cells and
om cul u es in ec ed wi h HSV-1 a MOIs o 0.001, 0.01, 0.1, 1 and 10
a he indica ed ime poin s. Samples we e esol ed on SDS-PAGE and
ans e ed on o ni ocellulose il e s. The s eady-s a e le els o gD,
Bax-α, Bax-β and p63 we e analyzed by Wes e n blo assay. To de e -
mine p o ein le els in HSV-1-in ec ed cells, band in ensi ies we e quan-
i ied by use o he ImageQuan so wa e. The numbe s indica e he
ela i e quan i ies o each band, no malized o he con ol cells a each
ime poin . Lanes 1, 6, 11, 16, 21 and 26, mock-in ec ed cells; lanes 2-5,
7-10, 12-15, 17-20, 22-25 and 27-30, HSV-1-in ec ed cul u es. The e-
sul s a e ep esen a i e o h ee independen expe imen s.
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ΔN o ms. The ΔNp63-speci ic an ibody p epa a ion
de ec ed he 68 kDa p63 iso o m in he mock-in ec ed
SIRC cells, bu ailed o ecognize he 51-62 kDa p63 iso-
o m in he cul u es in ec ed wi h HSV-1 a an MOI o 10
o 24 hpi (Fig 5). These esul s clea ly e eal ha he 68
kDa p63 p o ein de ec ed in he mock-in ec ed SIRC cells
is ΔNp63α, while he 51-62 kDa p63 iso o m de ec ed in
HSV-1-in ec ed cul u es is TAp63γ.
Toge he , hese esul s indica e ha HSV-1 modula es
he exp ession pa e ns o Bax and p63. The le el o
ΔNp63α was dec eased, while he exp essions o Bax-β
and TAp63γ we e highly inc eased in he HSV-1-in ec ed
SIRC cells.
HSV-1-media ed TAp63γ exp ession equi es i al DNA
eplica ion
To in es iga e he basis o he HSV-1-induced inc ease o
he TAp63γ le el, SIRC cells we e in ec ed in he p esence
o absence o he i al DNA eplica ion inhibi o ACG.
The cells we e analyzed o he p esence o HSV gD,
ΔNp63α, TAp63γ and Bax-β. The low le el o he la e
p o ein gD exp ession in SIRC samples ea ed wi h 50 o
10 μg/ml ACG indica ed ha he d ug ea men e i-
cien ly inhibi ed i al DNA eplica ion (Fig. 6; lanes 2 and
3).
The Bax-β p o ein le els in he HSV-1-in ec ed SIRC
cells ea ed wi h 50, 10 and 1 μg/ml ACG we e g ea ly
dec eased ( he ela i e quan i ies o Bax-β in cells
in ec ed a an MOI o 10 we e 0.12, 0.15 and 0.21, espec-
i ely) (Fig. 6; lanes 2-4).
The TAp63γ p o ein le els in he HSV-1-in ec ed SIRC
cells ea ed wi h 50 and 10 μg/ml ACG we e g ea ly
dec eased ( he ela i e quan i ies o TAp63γ in cells
in ec ed a an MOI o 10 we e 0.11 and 0.19) (Fig. 6; lanes
2 and 3). The exp ession o he TAp63γ iso o m in he
HSV-1-in ec ed cul u es ea ed wi h 1 μg/ml ACG was
down egula ed ( he ela i e quan i y o TAp63γ in SIRC
cells in ec ed a an MOI o 10 was 0.24) (Fig. 6; lane 4).
Discussion
This s udy, aiming o e alua e he ole o p63 in he
pa hogenic mechanisms o he pe ic ocula su ace dis-
ease, e ealed he p esence o HSV-1 gD p o ein and a
Figu e 6 The HSV-1-media ed TAp63γ exp ession equi es i al
DNA eplica ion. SIRC cells we e in ec ed wi h he KOS s ain o HSV-
1 a an MOI o 10 and main ained o 24 h in he absence o in he p es-
ence o Acyclo i (ACG). To de e mine he dependence o he TAp63γ
exp ession on HSV-1 DNA eplica ion, he le els o gD, Bax and p63
we e de e mined by Wes e n blo assay. To de e mine p o ein le els in
HSV-1-in ec ed cells, band in ensi ies we e quan i ied by densi ome ic
analysis wi h he Imagequan so wa e. The numbe s indica e he ela-
i e quan i ies o each band, no malized o he con ol cells a each
ime poin . Lane 1: HSV-1-in ec ed cells incuba ed in he absence o
ACG; lanes 2-4: HSV-1-in ec ed cells incuba ed in he p esence o ACG.
The esul s a e ep esen a i e o h ee independen expe imen s.
Figu e 5 Se ological iden i ica ion o he p63 iso o ms exp essed
in HSV-1-in ec ed SIRC cells. The le els o di e en p63 iso o ms
we e de ec ed a 24 hpi in mock-in ec ed and HSV-1-in ec ed SIRC
cells by Wes e n blo analysis, using an an ibody p epa a ion ha ec-
ognizes all o he a ious p63 iso o ms (lanes 1 and 2) and a ΔN-iso-
o m-speci ic immunoglobulin (lanes 3-7).

O osz e al. Jou nal o Biomedical Science 2010, 17:47
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s ong cy opa hic e ec in he HSV-1-in ec ed abbi co -
neal cell line (SIRC) (Figs 2, 3, 4). Ou da a ha e also indi-
ca ed ha apop osis plays an impo an ole in he demise
o SIRC cells in ec ed wi h HSV-1 (Fig. 3). These da a a e
in ull ag eemen wi h p e ious indings demons a ing
ha HSV-1 has he po en ial o elici a ious o ms o cell
dea h, including nec osis, apop osis, anoikis and
au ophagy [29-35].
Compelling e idence has accumula ed ha he a ious
p63 iso o ms play pi o al oles in se e al physiological
and pa hological p ocesses o he ocula su ace [13-19].
The ΔN and TA p63 subclasses ope a e in a conce ed
ashion o main ain he p oli e a i e po en ial o he ocu-
la su ace epi helia and o con ol he p ocesses o di e -
en ia ion and egene a ion in he conjunc i a and co nea
[15,19]. The ocula su ace may be exposed o ha m ul
en i onmen al s imuli, such as ul a iole exposu e, and
may also unc ion as an en y si e o a wide a ay o
human pa hogenic mic oo ganisms. By dis u bing he
delica e balance be ween he p o-su i al ΔN and he
p o-apop o ic TA iso o ms, s ess signals ha al e he
exp ession o p63 may cause p o ound al e a ions in he
iabili y o he ocula cells and in he issue homeos asis
o he ocula su ace. As a s ep in ou in es iga ions o he
unde lying molecula e en s implica ed in HSV-1-
induced ocula cy opa hogenici y, we ocused on he ole
o p63 in he SIRC cell line. Ou expe imen s e ealed he
cons i u i e exp ession o ΔNp63α in he mock-in ec ed
SIRC cells (Fig. 4). In e es ingly, we obse ed an imp es-
si e educ ion in he le el o he ΔNp63α and a d ama ic
ise in he le el o TAp63γ ollowing in ec ion wi h HSV-
1 (Fig. 4). The kine ics o HSV-1 eplica ion and he le el
o TAp63γ exp ession co ela ed s ic ly (Fig. 4). No e-
wo hy p e ious s udies aise he possibili y ha HSV-1
may al e he exp ession o p63 ia mul iple mechanisms
[36-45]. Ce ain i al p o eins may ha e he po en ial o
al e he ansc ip ion o p63 o o a ec he s abili y and
ac i i y o he p63 iso o ms ia he induc ion o hei
pos ansla ional modi ica ions [36-44]. The i ion-asso-
cia ed hos shu o p o ein [( hs), also known as UL41],
which causes he deg ada ion o cellula and i al RNA
[36,37], may e oke a dec ease in he le el o ΔNp63α
mRNA. The α- ans-inducing ac o [(α-TIF), also known
as VP16 o UL48], which s imula es he ansc ip ion o
IE genes ia cellula ansc ip ion ac o s, such as he
POU homeodomain p o ein Oc -1 (whe e Oc s ands o
oc ame binding p o ein) and he hos cell ac o [38-40],
may elici an inc ease in he le el o TAp63γ. The in ec ed
cell p o ein (ICP) 0, which con ols he s abili y o cellula
p o eins and leads o he dis up ion o p omyelocy ic leu-
kemia (PML) nuclea bodies [also known as PODs (PML
oncogenic domains) and ND10 (nuclea domain 10)] [41-
44], may dys egula e he exp ession pa e n o p63. How-
e e , in e es ing s udies ha e demons a ed ha he epli-
ca ion o HSV-1 DNA ac i a es he a axia eleangiec asia
mu a ed (ATM)-dependen signaling pa hway implica ed
in he cellula DNA damage esponse (DDR) [45]. Since
TAp63 iso o ms ha e been shown o ope a e as impo -
an downs eam media o s o DDR [46-48], i is concei -
able ha he dys egula ion o p63 exp ession obse ed in
HSV-1 in ec ed SIRC cells is a esul o he ac i a ion o
DDR e oked by i al eplica ion. Ou expe imen s ha e
shown ha he i al DNA eplica ion inhibi o ACG
comple ely abolished he HSV-1-media ed induc ion o
TAp63γ in SIRC cells, indica ing ha eplica ion o i al
DNA is necessa y o he accumula ion o TAp63γ (Fig.
6). This obse a ion s ongly suppo s he iew ha he
dys egula ion o p63 exp ession depends on he cellula
DDR, bu does no exclude he ole o HSV-1-encoded
p o eins. Thus, addi ional s udies a e equi ed o eluci-
da e he po en ial con ibu ions o hs, α-TIF, ICP0 and
o he i al p o eins o he de elopmen o he HSV-1-
media ed dys egula ion o p63 exp ession. Ou da a u -
he demons a ed ha HSV-1-in ec ed SIRC cells display
dec eased iabili y and an inc eased apop o ic a e (Fig.
3). Toge he , hese esul s sugges ha he al e ed pa e n
o p63 exp ession obse ed in HSV-1-in ec ed SIRC cells
may ep esen a mechanism by which his i us pe u bs
he unc ions o he co neal epi helial cells and leads o
hei demise.
In line wi h hese da a, we nex in es iga ed he exp es-
sion o Bax, which is known o be up egula ed by TAp63α
and TAp63γ [10,11]. P e ious s udies ha e demons a ed
he exis ence o se e al Bax iso o ms [49]. I is well docu-
men ed ha Bax-α is a cen al componen o apop osis
induc ion [50]. In esponse o apop o ic s imuli, Bax-α
becomes ac i a ed, ansloca es o he mi ochond ia and
igge s he elease o cy och ome c and caspase-9, which
in u n esul s in he i e e sible execu ion o he apop-
o ic p og am [51]. I has been epo ed ha he Bax-β
p o ein is exp essed cons i u i ely in se e al human cell
ypes, and i s le el is con olled by p o easomal deg ada-
ion [52]. Va ious s esso s inhibi ubiqui ina ion o he
Bax-β p o ein and he eby p e en i s p o easomal deg a-
da ion, leading o he accumula ion o his Bax iso o m
[52]. Simila ly o Bax-α, Bax-β has he capabili y o ig-
ge apop osis ia he mi ochond ial pa hway [52,53].
Mo eo e , Bax-β associa es wi h and p omo es Bax-α
ac i a ion [53]. Ou expe imen s e ealed no cons i u i e
exp ession o any o he Bax iso o ms in he mock-
in ec ed SIRC cells (Fig. 4). In e es ingly, we obse ed a
d ama ic ise in he le el o Bax-β in HSV-1-in ec ed cul-
u es (Fig. 4). Following he demons a ion o an al e ed
Bax exp ession pa e n in SIRC cells, we pos ula e an
impo an ole o Bax-β in he apop o ic esponsi eness
o co neal epi helial cells in ec ed wi h HSV-1. O he
in e es ing ecen da a ha e p o ed ha HSVs encode
ubiqui ina ing and deubiqui ina ing enzymes, which can
O osz e al. Jou nal o Biomedical Science 2010, 17:47
h p://www.jbiomedsci.com/con en /17/1/47
Page 7 o 8
modi y he ubiqui ina ion s a us o bo h i al and hos
cell p o eins [54,55]. In iew o hese obse a ions, i is
easonable o in e ha he Bax-β p o ein may be a no el
a ge o HSV-1-media ed deubiqui ina ing e en s. How-
e e , he p ecise molecula mechanisms esponsible o
s abiliza ion o he Bax-β p o ein in HSV-1-in ec ed cells
emain o be elucida ed.
Conclusions
O e all, his s udy demons a es ha he KOS s ain o
HSV-1 modula es he pa e ns o p63 and Bax exp ession
in he SIRC cell line. These da a may bea on he pa ho-
genic mechanisms o ocula diseases caused by HSV-1, as
p63 and Bax iso o ms play a pi o al ole in he main e-
nance o he ocula su ace in eg i y.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Au ho s' con ibu ions
LO designed and pe o med mos expe imen s, and d a ed he manusc ip ,
ÉG helped o design expe imen s and edi ed he manusc ip , LK helped o
design expe imen s, in e p e ed he esul s and e ised he manusc ip , YM
helped o design he expe imen s, in e p e ed he esul s and e ised he man-
usc ip , AF helped o design expe imen s and edi ed he manusc ip , KM con-
cei ed o he s udy, pe o med esea ch and e ised he manusc ip . All
au ho s ead and app o ed he inal manusc ip .
Acknowledgemen s
We hank Gyöngyi Áb ahám o expe echnical assis ance. This s udy was
suppo ed by g an s OTKA/T043144 om he Hunga ian Scien i ic Resea ch
Fund and ETT/398/2003 om he Hunga ian Minis y o Heal h, Social and
Family A ai s.
Au ho De ails
1Depa men o Medical Mic obiology and Immunobiology, Uni e si y o
Szeged, Dóm é 10, H-6720 Szeged, Hunga y, 2Depa men o
Oph halmology, Uni e si y o Szeged, Ko ányi aso 10-11, H-6720 Szeged,
Hunga y, 3Depa men o De ma ology and Alle gology, Uni e si y o Szeged,
Ko ányi aso 6, H-6720 Szeged, Hunga y, 4De ma ological Resea ch G oup o
he Hunga ian Academy o Sciences, Ko ányi aso 6, H-6720 Szeged, Hunga y
and 5Depa men o Oph halmology, Uni e si y o Deb ecen, Nagye dei kö ú
98, H-4032 Deb ecen, Hunga y
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This a icle is a ailable om: h p://www.jbiomedsci.com/con en /17/1/47© 2010 O osz e al; licensee BioMed Cen al L d. 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 (h p://c ea i ecommons.o g/licenses/by/2.0), 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 wo k is p ope ly ci ed.Jou nal o Biomedical Science 2010, 17:47
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Ci e his a icle as: O osz e al., In ol emen o p63 in he he pes simplex
i us-1-induced demise o co neal cells Jou nal o Biomedical Science 2010,
17:47