Squamous cell carcinoma antigen 1 and 2 expression in cultured normal peripheral blood mononuclear cells and in vulvar squamous cell carcinoma

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1 Tumor Biol. (2010) 31: DOI /s x RESEARCH ARTICLE Squmous ell rinom ntigen 1 n 2 expression in ulture norml peripherl loo mononuler ells n in vulvr squmous ell rinom Mglen Chehlinsk & Mglen Kowlewsk & Eyt Brzosk-Wojtowiz & Jku Rziszewski & Konr Ptszynski & Jnusz Rys & Jnin Kminsk & Roslw Nowk Reeive: 13 April 2010 /Aepte: 10 June 2010 /Pulishe online: 1 July 2010 # The Author(s) This rtile is pulishe with open ess t Springerlink.om Astrt Squmous ell rinom ntigen (SCCA) is expresse in norml squmous ell epitheli n in squmous ell rinoms (SCC). Two nerly ientil genes enoe the inhiitory serpins SCCA1 (SERPINB3) n SCCA2 (SERPINB4). Serum levels of SCCA re elevte in ptients with enign skin iseses n in ptients with SCC. SCCA, use for the monitoring of SCC ptients, presents no stisftory ignosti speifiity. As we hve shown previously, the reverse trnsription polymerse hin retion (RT-PCR)-se SCCA messenger RNA (mrna) testing ime t eteting isseminte ner ells my e hmpere y the flse-positive results ue to SCCA expression in tivte peripherl loo mononuler ells (PBMC). The im of this stuy ws to M. Chehlinsk (*) Deprtment of Immunology, Mri Skloowsk-Curie Memoril Cner Centre n Institute of Onology, Roentgen 5, Wrsw, Poln e-mil: heh@oi.ww.pl M. Kowlewsk : R. Nowk Deprtment of Moleulr Biology, Mri Skloowsk-Curie Memoril Cner Centre n Institute of Onology, Roentgen 5, Wrsw, Poln E. Brzosk-Wojtowiz Deprtment of Cytology, Institute of Zoology, Biology Fulty, Wrsw University, Mieznikow 1, Wrsw, Poln J. Rziszewski Deprtment of Brhytherpy, Mri Skloowsk-Curie Memoril Cner Centre n Institute of Onology, Roentgen 5, Wrsw, Poln ssess the expression of SCCA t mrna n protein levels in ulture norml PBMC, ompre to tht in vulvr SCC (VSCC) smples. High SCCA onentrtions were foun in vulvr tumours n in metstti lymph noes, while negtive inguinl lymph noes from the sme ptients often presente signifintly less SCCA. In norml tivte PBMC, the level of SCCA protein ws the lowest. At the mrna level SCCA ws etetle in norml PBMC even in ultures with no mitogen stimultion, ut only y the neste RT-PCR, ontrry to VSCC smples foun to e SCCA positive lrey in one-step PCR. Both SCCA1 n SCCA2 trnsripts were present in ulture PBMC; SCCA1 ws expresse t higher level thn SCCA2. In onlusion, oth SCCA forms re etetle in norml K. Ptszynski Deprtment of Pthology, Mri Skloowsk-Curie Memoril Cner Centre n Institute of Onology, Roentgen 5, Wrsw, Poln J. Rys Deprtment of Pthology, Mri Skloowsk-Curie Memoril Cner Centre n Institute of Onology, Grnrsk 11, Krkow, Poln J. Kminsk Deprtment of Tumour Mrkers, Mri Skloowsk-Curie Memoril Cner Centre n Institute of Onology, Roentgen 5, Wrsw, Poln

2 560 Tumor Biol. (2010) 31: PBMC ulture in vitro. SCCA expression level in norml PBMC is muh lower thn in the squmous epitheliumerive ells. In VSCC, in ition to tumour itself, metstti lymph noes seem lso to e potentil soure of serum SCCA. Keywors SCC. SERPINB3. SERPINB4. Tumour mrker. PBMC. Vulvr rinom Introution Squmous ell rinom ntigen (SCCA), first isovere in the squmous ell rinom of the ervix [1], is expresse in ifferent squmous ell rinoms (SCC) n in norml squmous ell epitheli. SCCA elongs to the fmily of inhiitory serine protese inhiitors (serpins), involve in poptosis, ell migrtion n invsiveness [2]. Elevte levels of serum SCCA proteins re ommon in ptients with enign, inflmmtory skin iseses, suh s psorisis n topi ermtitis, s well s in ptients with squmous ell rinoms, espeilly of the ervix, he n nek, lung, oesophgus n vulv, where SCCA onentrtions my orrelte with linil stge, histologil gre n tumour uren [1, 3 7]. Serum SCCA hs n inepenent prognosti vlue in ptients with ervil rinom n with he n nek mlignnies [6, 8] n is use for the ignosis n monitoring of ptients with SCC [9]. There re two SCCA proteins, enoe y two, nerly ientil genes, SCCA1 (SERPINB3) n SCCA2 (SERPINB4). SCCA1 n SCCA2 proteins, lthough they present 92% mino i sequene homology n trget ifferent proteses. The neutrl SCCA1 inhiits ppine-like lysosoml ysteine proteses, thepsine S, L n K, n the ii SCCA2 inhiits the serine proteses, thepsine G n mst ell hymse [10, 11]. Both SCCA proteins re ololise in norml n mlignnt squmous epithelil ells, ut the ominting serum form in ptients with SCC is SCCA2 [12 14]. Elevte SCCA2/SCCA1 messenger RNA (mrna) rtio in primry SCC of the he n nek n of ervix ws foun to e n inepenent preitor of tumour reurrene [15, 16]. SCCA is lso regre s moleulr tumour mrker for isseminte SCC ell etetion [17]. However, s we hve previously shown while stuying the question of flse-positive reverse trnsription polymerse hin retion (RT-PCR) results of isseminte ner ell etetion [18], there is n expression of numer of the so-lle tumourspeifi moleulr mrkers, inluing SCCA, in tivte peripherl loo mononuler ells (PBMC). Here, we ime to stuy the totl SCCA s well s SCCA1 n SCCA2 expression t oth the mrna n protein levels, in norml unstimulte n PHA-stimulte PBMC, n to ompre the otine totl SCCA protein expression to tht in vulvr squmous ell rinom (VSCC), one of mny types of SCCs. Mteril n methos PBMC n ell lines PBMC of six helthy onors were otine y stnr Fioll-Pque (Phrmi) grient entrifugtion; smples of unulture ells were tken, n the remining ells were ulture in stnr meium supplemente with 10% fetl lf serum (FCS) n gentmyin, for 1 y without mitogen, n in prlell, for 1 6 ys in the presene of PHA (1 μg/ml). Cultures were entrifuge, superntnts were ollete n frozen, ells were wshe twie in PBS n the ell pellet ws kept frozen t 70 C until RNA ws isolte. Squmous epithelil ell lines, the epiermoi vulvr rinom ell line, A431 (Deutshe Smmlung von Zellkulturen un Mikroorgnismen, Brunshweig, Germny), the humn ervil rinom ell line, SKG- III (RINKEN BRC, Ntionl Bio-Resoure Projet of the MEXT, Jpn), the suprglotti lrynx rinom ell line, UT-SCC-8 (The University of Turku, Finln), n the humn kertinoyte ell line, HCt [19], s well s the ervix enorinom ell line, HeL (Wrsw Meil University, Wrsw Poln), were ulture t 37 C, 5% CO 2 in the stnr mei supplemente with 10% FCS n gentmyin. Post-surgil smples from vulvr rinom ptients We stuie the smples otine from surgil speimens of 41 ptients trete in the Mri Skloowsk-Curie Memoril Cner Center n Institute of Onology for VSCC (pt1-2, N0-2, M0). All ptients gve their informe onsent. Smples inluing 39 tumours, 15 regionl lymph noes n 36 uninvolve jent tissues were snp-frozen n store t 70 C until they were pulverise on liqui nitrogen, using Miroismemrtor II (B Brun Bioteh Interntionl, Melsungen, Germny). The uninvolve tissues omprise ross-setions of skin next to the surgil mrgin. Histologilly, they were ompose of ermis n the epierml lyer. RNA isoltion n RT-PCR Totl RNA ws isolte from PBMC t eh time point (0, efore ulture; unstimulte 1-y ultures; n PHAstimulte 1 6-y ultures ) n from pulverise postsurgil smples from VSCC ptients with the use of

3 Tumor Biol. (2010) 31: Quigen RNesy kits. The quntity n qulity of the RNA tht ws otine were exmine y spetrophotometry (Nnorop, Thermosientifi) n y enturing gel eletrophoresis with ethiium romie. Two mirogrms of the totl RNA ws reverse trnsrie with rnom hexmer primers, in the finl volume of 20 μl, using SuperSript reverse trnsriptse (Invitrogen), oring to the mnufturer s instrutions. The qulity of omplementry DNA preprtions ws ontrolle y the PCR of DNA polymerse β, s esrie previously [18]. Totl SCCA, SCCA1 n SSCA2 expression ws nlyse in PBMC y the neste RT-PCR with speifi primers. Tissue smples otine from VSCC ptients were exmine for totl SCCA expression y one-step RT-PCR. Mesurements of SCCA protein expression Totl ell protein ws extrte from unstimulte n stimulte PBMC of four norml onors n from pulverise post-surgil smples of seven ptients with vulvr ner, oring to the proeure esrie elsewhere [13]. Totl SCCA expression in the protein extrts of PBMC n vulvr tumours, the onentrtions of SCCA (totl) in ulture mei n 22 ptients ser were mesure using the Arhitet SCCA kit of Aott, n SCCA in protein extrts were expresse in nnogrm per milligrm of ell protein (CP), while in the ser n ulture mei in nnogrm per millilitre. Immunoytohemil stining ws performe using primry nti-scca1, nti-scca2 MA (Snt Cruz, 8H11 n 10C12, respetively) n seonry nti-mouse IgG rit ntioy Alex 633 onjugte (Invitrogen). Nulei were ounterstine with hromomyin A 3 (Sigm), n the results were imge with onfol mirosope (Axiovert 100 M, Zeiss) n LSM 510 META pplition. Immunohistohemil stining ws performe with nti- SCCA1 (8H11), nti-scca2 (10C12) n nti-totl SCCA (B-9) MAs (Snt Cruz). Nulei were ounterstine with hemtoxylin n eosin, n the results were imge uner light mirosope. Results The speifi primers for totl SCCA, SCCA1 n SCCA2 mplifie the orret sequenes, s onfirme y sequening. As shown y the neste RT-PCR, totl SCCA ws expresse in mitogen-stimulte PBMC, where it persiste for ll 6 ys of ulture, s well s in unstimulte PBMC following 1-y ulture without mitogen (Fig. 1 n Tle 1). SCCA ws unetetle in the freshly isolte PBMC /PHA 2/PHA 3/PHA 4/PHA 5/PHA 6/PHA NEG (No Templte) Donor I 0 1 1/PHA 2/PHA 3/PHA 4/PHA 5/PHA 6/PHA Jurkt NEG (No Templte) DNA Donor II Fig. 1 Expression of totl SCCA mrna in PBMC from onor I (0 efore ulture, 1 unstimulte 1-y ulture n PHA stimulte 1 6 y ultures) The SCCA1 n SCCA2 genes usully o-expresse in PBMC, n this ws lso oserve in ultures with no mitogen stimultion. In PBMC from onor I, the level of SCCA1 expression ws onstnt up to 3 ys of ulture, ut in the susequent 3 ys, PBMC presente no SCCA1 expression, in ontrst to SCCA2, whih ws etetle throughout the whole ulture perio (Fig. 2 n Tle 1). The level of SCCA2 expression ssesse semiquntittively in 1-y ultures ws foun to e lower thn tht of SCCA1, in oth stimulte n unstimulte PBMC of onor I (Fig. 3). The totl SCCA ws foun to e onsequently expresse in PBMC of ll onors throughout the ulture, while the SCCA1 n SCCA2 gene expression, lthough foun to e expresse in ll onors, ws ientifie in ifferent time points in ifferent onors (Tle 1). The SCCA mrna expression in post-surgil smples from ptients with VSCC ws lso exmine. Alrey in one-step PCR, ll 39 tumour smples were SCCA positive, while the squmous ell vulvr rinom ell line A431 ws SCCA negtive (A431 ws positive in two-step PCR). The SCCA trnsripts were lso etete in 31 out of 36 uninvolve jent tissues (86%).

4 562 Tumor Biol. (2010) 31: Tle 1 Totl SCCA, SCCA1 n SCCA2 expression, ssesse y the neste RT-PCR Totl SCCA SCCA1 SCCA2 Donor I II II II III III IV V VI I II II III IV V VI I II II III III IV V VI Norml PBMC Dys of ulture 0 n.. n.. n.. n.. n n n.. n.. n n.. n.. n n.. n.. n.. 1 /PHA n n n n /PHA n n.. n.. n n.. n.. n.. +/ n.. n.. n.. 3 /PHA n n.. n.. n n.. n.. n.. +/ n.. n.. n.. 4 /PHA n n.. n.. n.. n.. n.. n.. +/ n.. n.. n.. 5 /PHA n.. n.. n.. n.. n.. n.. +/ n.. n.. n.. 6 /PHA n n.. n.. + n.. n.. + n.. n.. In onors II n III repete ssessments were performe (+) present, ( ) sent, (+/ ) tre expression, n.. not etermine The SCCA onentrtion in the protein extrt of tivte PBMC ws foun to reh 2.7 ng/mg of CP (men, 2.15 ng/mg; Tle 2). In the post-surgil smples from VSCC ptients, SCCA onentrtions were two to four orers of mgnitue higher thn those in ulture PBMC. The SCCA protein onentrtions were usully higher in the tumour thn in the uninvolve jent tissue (Tle 2). Metstti inguinl lymph noes expresse higher levels of SCCA protein thn uninvolve inguinl lymph noes erive from the sme ptients. Interestingly, SCCA1 SCCA2 Negtive Dy 0 Dy 1 Unstimulte Dy 1 PHA Dy 2 PHA Dy 3 PHA Dy 4 PHA Dy 5 PHA Dy 6 PHA DNA ler Dy 0 Dy 1 Unstimulte Dy 1 PHA Dy 2 PHA Dy 3 PHA Dy 4 PHA Dy 5 PHA Dy 6 PHA Negtive Fig. 2 SCCA1 n SCCA2 mrna expression kinetis in stimulte PBMC from onor I in ptients 12 n 30, the lymph noes tht h een lssifie histpthologilly s uninvolve (Tle 2) presente reltively high SCCA levels. This might suggest tht these lymph noes i ontin mirometstses. Immunoytohemil nlysis uner onfol mirosope hs shown tres of SCCA 1 n 2 proteins in ulture PBMC (Fig. 4). As shown in onor III, the SCCA1 n 2 expression t the protein level ws etete in PBMC lter fter stimultion thn t the mrna level (Tle 3). The immunohistohemil nlysis of tumour n inguinl lymph noe from ptient with VSCC onfirme the eviently stronger SCCA stining in the metstti lymph noe thn in the SCC smple (Fig. 5) n showe tht not only ner ells express SCCA protein (Tle 4). SCCA seretion ws exmine in ell ultures. SCCA ws unetetle in the spent mei from PBMC ultures, s well s from HCt kertinoyte ell line (<0.3 ng/ml), while it ws serete in reltively high mounts y the squmous ervil ner ell line, SKG-III (8 ng/ml), known for high SCCA expression level. The mein SCCA level in the ser of 22 ptients with erly VSCC ws 1.35 ng/ml ( ). Only four (18%) of SCCA1 SCCA2 SCCA1 SCCA2 Unstimulte PBMC fter 1 y ulture PHA-stimulte PBMC SCCA1/SCCA2 rtio Fig. 3 Semiquntittive ssessment of SCCA1/SCCA2 expression rtio in PBMC

5 Tumor Biol. (2010) 31: Tle 2 SCCA onentrtions (Aott IMX SCCA ssy) in the protein extrts of norml unstimulte (y 0) n stimulte (y 3) peripherl loo mononuler ells (PBMC) n of tissue smples s well s in the ser from ptients with VSCC Donor numer Ptient numer Helthy onor PBMC I II III IV Unstimulte Stimulte Smples of VSCC Tumour Ajent uninvolve tissue Metstti lymph noe x x Uninvolve lymph noe Serum n. 0.9 n.. n Conentrtions of SCCA in the ells n post-surgil smples re presente in nnogrm per milligrm of ell protein n in the ser in nnogrm per millilitre of serum x no metstti noes foun, n.. not etermine More thn one lymph noe of this ptient ws teste those ptients h elevte SCCA levels, n three h SCCA t the ut-off level (2 ng/ml). Disussion We were the first to show the SCCA expression to e inuile in norml PBMC [18]. Here, we stuie the totl SCCA expression long with the expression of its ii n neutrl forms in more etil. We onfirme our erlier results [18] n those of Cttelpe [12], who stuie ifferent norml tissues, tht SCCA mrna is not expresse in unulture norml PBMC. However, s we showe here, norml, tivte PBMCs express SCCA1 n SCCA2 t oth the mrna n protein level. The two SCCA forms re often ientifie in ifferent time points following stimultion, oth in protein n mrna stuies. Interestingly, lso unstimulte PBMCs following ulture in the presene of FCS were foun to express SCCA1 n SCCA2 trnripts. In ition, oth in unstimulte (ulture for 1 y without mitogen) n in stimulte norml PBMC, the SCCA1 expression mesure t the mrna level ws higher thn the SCCA2 expression. Our semiquntittive ssessment of the reltive SCCA1 n SCCA2 expression revele tht PBMC UT SCC 8 HeL HeL NEG SCCA1 Negtive Control SCCA2 SCCA2 Dy 0 Fig. 4 SCCA1 n SCCA2 expression (green) in stimulte PBMC n UT-SCC-8 n HeL ell lines. Confol mirosope imges. Chromomyin-stine nulei (re), SCCA 1 or SCCA 2 fluoresene, trnsmitte light imges, n overly of pnels on, illustrting the ytoplsmi fluoresene

6 564 Tumor Biol. (2010) 31: Tle 3 A omprison of SCCA1 n SCCA2 expression in the peripherl loo mononuler ells (PBMC) mrna expression ws evlute y neste RT-PCR, protein expression ws estimte y immunoytohemistry (+) present, ( ) sent, (+/ ) tre expression Donor III Norml PBMC SCCA1 mrna Anti-SCCA1 SCCA2 mrna Anti-SCCA /PHA + +/ 2/PHA +/ + 3/PHA 4/PHA + 5/PHA +/ + 6/PHA the SCCA1/SCCA2 rtio inrese 1.5 fol (1.54:2.38) upon PBMC stimultion. Similrly, squmous epitheliumerive ells, suh s norml kertinoytes, norml nonkertinoyte ells, ervil squmous ell rinom n enorinom ells, hve een shown to express higher mrnalevelsofscca1thnofscca2[20]. However, in squmous ell rinoms of he n nek, Stenmn et l. [15] oserve erese in SCCA1/SCCA2 expression rtio in mlignnt squmous ells; tht is, the SCCA1/ SCCA2 mrna rtios were lower in tumour tissue (mein rtio<5) thn in norml epithelium (mein rtio>10). Those rtios were lso lower in primry tumours tht evelope into reurrent isese n in reurrent seonry tumours thn in non-reurring primry tumours. Similrly, Hsu et l. [16] foun tht in the uterine, the SCCA1/SCCA2 mrna rtios were progressively erese in norml vs. ysplsti n ysplsti vs. ner ells. In ition, the erese SCCA1/SCCA2 rtio rrie higher risk for reurrene in erly-stge uterine ervil ner. The pulishe t on SCCA expression in norml humn tissues re inonsistent. In the squmous ells of norml epitheli, some uthors reporte the SCCA immunoretivity [21, 22], while others [23, 24] etete low or sent SCCA expression. The isrepnies might result from the ifferent ntioy speifiities n tissue origins. Ctltepe et l. [12] hve shown stronger SCCA1 n SCCA2 immunoretivity in norml skin over res of Fig. 5 Expression of totl SCCA n its forms (SCCA1 n SCCA2) in the prfin setions of VSCC n metstti lymph noe setions. Immunohistohemil stining (Snt Cruz MA) Negtive Control Vulvr SCC Metstti lymph noe totl SCCA

7 Tumor Biol. (2010) 31: Tle 4 Totl SCCA, SCCA1 n SCCA2 expression oring to ell types, ientifie y immunohistohemil exmintion of vulvr tumour n inguinl lymph noe setions T tumour, N inguinl lymph noe, 0 no suh ells ientifie, (+) present, ( ) sent Cell types Cners Grnuloyte Lymphoytes Plsmoytes Norml sl epithelil T N T N T N T N T N Totl SCCA SCCA SCCA inflmmtion. This oservtion inites tht the expression of SCCA in the skin n e inue y lol inflmmtion. Inee, SCCA hs een reognise s mrker of inflmmtory skin iseses [23, 25, 26]. Interestingly, in the squmous ell rinoms of the tongue, Ysumtsu et l. [27] oserve SCCA1 immunostining in the T-lymphoytes peripherl to ner ells n foun this stining, ut not SCCA1 expression in ner ells, to signifintly orrelte with elevte serum SCCA onentrtions. We showe here SCCA immunoretivity in vulvr tumour-ssoite grnuloytes. Thus, the soure of serum SCCA my not neessrily e ner ells ut other tumour-ssoite ells, suh s infiltrting immune ells. Elevte serum SCCA ssoite with severl nonneoplsti ermtologil iseses [23, 25] s well s with renl filure or lung iseses [28] support the possile role of ifferent non-neoplsti ells in SCCA relese. Furthermore, Vn er Sije et l. [29], who foun serum SCCA ssessment useful tool for the erly etetion of reurrent VSCC, lso oserve 25% of flse-positive (i.e. in ptients with no ner) SCCA results in ptients with enign skin isorers. In ition, elevte onentrtions of serum SCCA n/or C-retive protein were reporte to e inepenent unfvourle prognosti ftors in he n nek squmous ell rinoms [30], whih links SCCA with systemi symptoms of ner-relte inflmmtion. Tken together, these t suggest tht SCCA is not only mrker of squmous ell rinoms ut lso of inflmmtory onitions, n lthough high sensitivity of serum SCCA etermintion in ner ptients is generlly ppreite, t the sme time its limite speifiity is evient. Here, we lso exmine SCCA expression in VSCC ptients. The levels of SCCA protein n mrna inue in norml PBMC were severl orers of mgnitue lower thn those foun in the vulvr tumour ells n in the ells of tumour-jent tissues n lymph noes. However, high levels of SCCA in the lol tumour miroenvironment n regionl metstti sites in ptients with VSCC seem not to imply elevte serum SCCA levels, s few of those ptients h serum SCCA onentrtions ove the norml level. In ptients with erly stge VSCC, onsistently with our t, other reserhers hve shown men serum SCCA level to e elow the ut-off vlue [4]. Serum SCCA levels were foun not to e ssoite with lymph noe involvement [4, 29, 31]. In vne VSCC (FIGO stge IV), less thn 40% of ptients present elevte serum SCCA onentrtions [4, 29]. The origin of serum SCCA in ptients with SCC remins n open question. Uemur et l. [13] hveshownthe lk of SCCA seretion y the SCC ells, ut other stuies ontrite this result [32, 33]. Our nlysis of SCCA onentrtions in the spent mei of stimulte PBMC, n of the squmous epitheli-erive ells of the HCt n SKG-III ervil ner ell lines, showe SCCA in the ulture mei of SKG-III ells ut not of PBMC or HCt ells. Thus, SCCA ientifie in oy fluis my either e serete y the ells n/or my erive from ying ells. In ition, SCCA expression n seretion is inuile y the Th2 ytokines, IL-13 n IL-4 in norml squmous epithelil ells [26, 33 35]. The Th1 to Th2 shift is regre s typil of ner; still, only SCC ptients present elevte serum SCCA onentrtions. Therefore, we hypothesise tht n inrese in the serum SCCA levels in ptients with squmous ell rinoms my e ssoite with SCCA inution y ytokines in norml squmous epithelil ells present in SCC miroenvironment. In summry, norml, tivte PBMCs express SCCA1 n SCCA2 t oth the mrna n protein level, ut the two forms of SCCA re not neessrily expresse onurrently. We oul not emonstrte tht ner-relte inflmmtory ells re potentilly signifint soure of irulting SCCA in VSCC. In vulvr SCC ptients, not only tumours ut lso metstti lymph noes re potentil soures of SCCA protein. We think tht the rise levels of serum SCCA oserve y some reserhers in the vne stges of vulvr rinom [36] my result from seonry hnges relte to the isese progression, suh s ltertions inue y metstti ner ells in their miroenvironment n/or n inrese in proinflmmtory ytokines. These hnges rete feek etween lol n systemi inflmmtion, phenomenon tht we hve isusse elsewhere [37]. The ext mehnisms of relesing of SCCA into the oy fluis remin to e resolve.

8 566 Tumor Biol. (2010) 31: Aknowlegements The uthors woul like to thnk Dr Młgorzt Jrmuż, Institute of Humn Genetis, Polish Aemy of Sienes, Poznn, Poln n Prof. Reir Grenmn, University of Turku, Finln, for proviing the UT-SCC-8 ell line, n Prof. Krzysztof Włorski, Wrsw Meil University, Wrsw, Poln, for proviing the HeL ell line. Open Aess This rtile is istriute uner the terms of the Cretive Commons Attriution Nonommeril Liense whih permits ny nonommeril use, istriution, n reproution in ny meium, provie the originl uthor(s) n soure re reite. Referenes 1. Kto H, Torigoe T. Rioimmunossy for tumor ntigen of humn ervil squmous ell rinom. Cner. 1977;40: Suminmi Y, Kishi F, Sekiguhi K, Kto H. Squmous ell rinom ntigen is new memer of the serine protese inhiitors. Biohem Biophys Res Commun. 1991;181: Torre GC. SCC ntigen in mlignnt n nonmlignnt squmous lesions. Tumour Biol. 1998;19: Hefler LA, Sliutz G, Leoolter S, Speiser P, Jour E, Reinthller A, et l. 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