13-cis Retinoic Acid Induces Apoptosis and Cell Cycle Arrest in Human SEB-1 Sebocytes

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1 ORIGINAL ARTILE See relted ommentry on pge is Retinoi Aid Indues Apoptosis nd ell yle Arrest in Humn SEB-1 Seoytes Amnd M. Nelson 1, Kthryn L. Gillilnd 1, Zhoyun ong 1 nd Dine M. Thioutot 1, Isotretinoin (13-is retinoi id ()) is the most potent inhiitor of seum prodution, key omponent in the pthophysiology of ne, yet its mehnism of tion remins lrgely unknown. The effets of, 9-is retinoi id (9-is RA), nd ll-trns retinoi id () on ell prolifertion, poptosis, nd ell yle proteins were exmined in SEB-1 seoytes nd kertinoytes. uses signifint dose-dependent nd time-dependent dereses in vile SEB-1 seoytes. A portion of this derese n e ttriuted to ell yle rrest s evidened y deresed DNA synthesis, inresed p1 protein expression, nd deresed ylin D1. Although not previously demonstrted in seoytes, we report tht indues poptosis in SEB-1 seoytes s shown y inresed Annexin V-FIT stining, inresed TUNEL stining, nd inresed leved spse 3 protein. Furthermore, the ility of to indue poptosis nnot e repitulted y 9-is RA or, nd it is not inhiited y the presene of retinoid id reeptor (RAR) pn-ntgonist AGN Tken together these dt indite tht uses ell yle rrest nd indues poptosis in SEB-1 seoytes y RAR-independent mehnism, whih ontriutes to its seosuppressive effet nd the resolution of ne. Journl of Investigtive Dermtology () 1, doi:1.13/sj.jid.579; pulished online 3 Mrh INTRODUTION Isotretinoin (13-is retinoi id ()) is the most potent inhiitor of seum prodution, key omponent in the pthophysiology of ne. It is the only retinoid tht drmtilly redues the size nd seretion of seeous glnds (Lndthler et l., 19; Struss et l., 19; Goldstein et l., 19). Despite the ft tht isotretinoin is extremely effetive ginst ne, surprisingly little is known regrding its moleulr mehnism of tion lthough dvnes re eing mde in this re. This unique retinoid hs een shown to ompetitively inhiit the 3-hydroxysteriod tivity of retinol dehydrogense leding to deresed ndrogen synthesis in vitro s well s inhiit the migrtion of polymorphonuler leukoytes into the skin supporting its role in the redution of inflmmtion tht is ssoited with ne (Wozel et l., 1991; Krlsson et l., 3). Numerous studies indite tht nd other retinoids ffet ell yle progression, differentition, poptosis, 1 The Jke Gittlen ner Reserh Foundtion, The Pennsylvni Stte University ollege of Mediine, Hershey, Pennsylvni, USA nd Deprtment of Dermtology, The Pennsylvni Stte University ollege of Mediine, Hershey, Pennsylvni, USA orrespondene: Dr Dine M. Thioutot, Deprtment of Dermtology, The Pennsylvni Stte University ollege of Mediine, HU1, 5 University Drive, Hershey, Pennsylvni 1733, USA. E-mil: dthioutot@psu.edu Arevitions: ANOVA, nlysis of vrine;, ll-trns retinoi id; 9-is RA, 9-is retinoi id;, 13-is retinoi id; NHEK, norml humn epiderml kertinoyte; RAR, retinoid id reeptor; RXR, retinoid X reeptor; TIG1, tzrotene-indued gene 1 Reeived 1 Septemer 5; revised 11 Ferury ; epted 13 Ferury ; pulished online 3 Mrh nd ell survivl in vriety of ell types inluding humn rest ners, orl squmous ell rinoms, lymphoytes, nd murine neurons (Pomponi et l., 199; Ginnini et l., 1997; Tom et l., 1997; riti et l., ; rndll et l., ; Ski et l., ). Like previous studies in other ell types, hs lso een shown to derese prolifertion of seoytes nd inhiit seoyte differentition s indited in histology speimens, primry seoytes, nd SZ95 immortlized humn seoytes (Dorn et l., 19; Jones et l., 19; Lndthler et l., 19; Struss et l., 19; Ridden et l., 199; Zououlis et l., 1991,, 1999). Although inresed levels of spse 3 were noted in SZ95 seoytes hours following tretment with nd inhiition of ell growth ws evident t 7 dys, other mrkers filed to indite tht SZ95 seoytes were undergoing poptosis (Zououlis et l., 1993; Wroel et l., 3). We hypothesized tht redues seoyte ell ounts vi ell yle rrest nd/or poptosis nd tht these effets might not e pprent within -hour tretment period. In this pper, we report tht fter nd 7 hours of tretment, ut not 9-is retinoi id (9-is RA) or ll-trns retinoi id (), inhiits growth nd indues poptosis in immortlized humn SEB-1 seoytes ut not in HT kertinoytes or norml humn epiderml kertinoytes (NHEK). Furthermore, the RAR pn ntgonist, AGN 19319, does not inhiit the poptosis indued y suggesting n RAR-independent poptoti mehnism. We hypothesize tht the ility of to indue ell yle rrest nd poptosis in seoytes ontriutes to the overll effet on suppression of seum nd improvement in ne. 17 Journl of Investigtive Dermtology (), Volume 1 & The Soiety for Investigtive Dermtology

2 Results exhiits more rpid onset of growth inhiition of SEB-1 seoytes ompred to 9-is RA or There is signifint dose-dependent derese in ell ount fter nd 7 hours of tretment with (Figure 1). At hours, onentrtions of.1,.5, nd 1 mm deresed ell ount y 19,, nd 3% respetively, when ompred to vehile (Po.5). After 7 hours ell numers deresed y 19, 3, nd 39% with onentrtions of.1 mm (Po.1),.5 mm (Po.1), nd 1 mm (Po.5) respetively. No signifint differenes in ell numer were noted t hours of tretment. Men±SEM vile ells 1 Men±SEM vile ells 1 Men±SEM vile ells μm hours..5 μm hours..5 μm 9-is RA hours Figure 1., 9-is RA, nd differentilly inhiit SEB-1 seoyte prolifertion. ( ) Time-dependent inhiition of SEB-1 ell prolifertion with individul retinoid ompounds. SEB-1 ells were ultured in the presene of ethnol vehile lone (.1% or less; ontrol).1,.5, or 1 mm onentrtions of, 9-is RA, or for,, or 7 hours. Atthed ells were olleted, stined with Trypn lue, nd vile ells ounted mnully y hemoytometer. Dt represent men7sem, n ¼ 1. Sttistil nlysis ws performed with ANOVA Two Ftor with Replition. Po.5, Po.1, nd Po.1. The effets of 9-is RA nd on SEB-1 seoyte ounts were noted eginning t 7 hours. Dereses of 39 nd 3% were noted with 9-is RA (.5 nd 1 mm, respetively) (Po.5) (Figure 1). (.1 nd 1 mm) signifintly deresed ell numer y 1 nd 37%, respetively (Po.5) (Figure 1). Overll, eh of these three retinoids deresed SEB-1 seoyte numers t 7 hours leit to vrying degrees ut effets were noted eginning t hours with. signifintly inhiits DNA synthesis in SEB-1 seoytes (.1,.5, nd 1 mm) eh signifintly deresed thymidine inorportion y pproximtely 3-fold t 7 hours (Po.1) (Figure ). No signifint hnges were noted t or hours. A 1.5-fold derese in 3 H thymidine Men±SEM.p.m. Men±SEM.p.m. Men±SEM.p.m μm hours..5 μm hours..5 μm 9-is RA hours Figure. inhiits DNA synthesis to greter extent thn 9-is RA or. ( ) SEB-1 seoytes were treted with ethnol vehile (ontrol) or.1,.5, 1 mm onentrtions of, 9-is RA, or for,, nd 7 hours. 1 mi 3 H thymidine ws dded to eh smple hours efore hrvesting. ells were wshed nd olleted for liquid sintilltion ounting. Dt represent men7sem, nx1. Sttistil nlysis ws performed with ANOVA Two Ftor with Replition. Po.5 nd Po

3 inorportion ws noted when ells were treted with 1 mm 9-is RA for hours (Figure ). in onentrtions of.5 nd 1 mm deresed thymidine inorportion y pproximtely 1. fold t nd 7 hours, respetively (Figure )., ut not 9-is RA or, inreses p1 levels in SEB-1 seoytes To further test the hypothesis tht hnges ell yle progression, expression of p1, ell yle inhiitor, ws exmined y Western lot. signifintly inresed p1 expression fter nd 7 hours of tretment (Figure 3). Speifilly, p1 levels inresed on verge.-fold nd 3.13-fold when ells were treted with.1 mm nd 1 mm, respetively, for hours (P ¼. nd.5). After 7 hours of tretment, ll onentrtions tested inresed p1 protein expression. used 1.7-,.7-, nd 3.1-fold inreses in p1 with.1, 1, nd 1 mm, respetively. No signifint differenes in p1 expression were noted t hours (dt not shown). When SEB-1 seoytes were treted with 9-is RA or in onentrtions of.1,.5, nd 1 mm, no signifint inreses in p1 protein were noted t or 7 hours (Figure 3 nd )., ut not 9-is RA or, dereses ylin D1 protein in SEB-1 seoytes To further explore the possiility tht indues G1 rrest in SEB-1 seoytes, ylin D1 protein ws exmined y Western lot. ylin D fmily memers re expressed nd funtion in ontrolling the progression from G1 to S phse in the ell yle (Bldin et l., 1993). Overexpression of ylin D1 shortens the durtion of G1 phse nd is rte limiting for phse progression (Quelle et l., 1993). Therefore, ylin D1 is likely ndidte to onfirm the tions of in inhiiting ell yle progression y influening the G1 to S phse trnsition. In SEB-1 seoytes, in onentrtions of.1, 1, nd 1 mm, signifintly derese ylin D1 protein t 7 hours (Figure 3d). No signifint effets of 13-is RA were noted t or hours (-hour dt not shown). 9-is RA or onentrtions of.1,.5, or 1 mm did not redue ylin D1 protein t 7 hours (dt not shown). indues poptosis in SEB-1 seoytes ut not in HT kertinoytes or NHEK To determine if the effet of on poptosis is elltype speifi, time ourse experiments were onduted in SEB-1 seoytes, HT kertinoytes, nd NHEK. In SEB-1 hours 7 hours hours 7 hours Reltive level p1±sem p μm. 1 μm hours. 1 μm 7 hours MM Reltive level p1±sem d p μm P =.7..5 μm..5 μm P =.5 p1..5 μm..5 μm ylin D1 β-. 1 μm. 1 μm MM 3 Reltive level p1±sem μm 9-is RA P =. Reltive level ylin D1±SEM μm Figure 3. inreses p1 nd dereses ylin D1 proteins. () SEB-1 ells were treted with.1, 1, nd 1 mm or vehile. ( nd ) Prllel experiments were performed with.1.5, or 1 mm onentrtions of 9-is RA nd. Blots were inuted with primry ntiodies to p1 nd -tin for loding ontrol normliztion nd nlyzed y densitometry. (d) SEB-1 ells were treted with.1, 1, nd 1 mm or vehile nd lots were inuted with primry ntiodies to ylin D1 nd -tin. Mgi Mrk XP (MM) indites nd size. Blots re representtive of minimum of three Western lots. Grphs represent normlized vlues reltive to vehile (ontrol) expression of minimum of three independent Western lots. Men7SEM. Po.5 nd P ¼.1. 1 Journl of Investigtive Dermtology (), Volume 1

4 seoytes, no signifint differenes in poptosis were noted in ells treted with for,,, or hours. A mrginl, yet signifint, inrese in the perentge of ells in erly poptosis ws noted in SEB-1 ells treted with.1 mm :.3.9% t hours nd from.19 to.% t 7 hours (Po.1 for eh time point). Signifint inreses in the perentge of ells in lte poptosis were noted t nd 7 hours with inresing onentrtions of (Figure, lte poptosis shown). Speifilly,.1 mm 13-is inresed the perentge of lte poptosis:. 5.% t hours nd % t 7 hours. t 1 mm onentrtion used inreses from 3. to 5.% nd from Men±SEM % of ells Men±SEM % of ells Men±SEM % of ells S hours hours. S hours hours. S SEB-1 HT NHEK hours 1 hours hours Figure. indues lte poptosis in SEB-1 seoytes ut not in HT kertinoytes or NHEK. () SEB-1 ells were treted with vehile (negtive ontrol), (.1 or 1 mm), or sturosporine (S) (positive ontrol) for indited times. () HT ells were treted with vehile, 13-is RA (.1 or 1 mm), or sturosporine (S) for the indited times. () NHEK ells were treted with vehile, (.1 or 1 mm), or sturosporine (S) for indited times. In ll experiments, ells were prepred ording to mnufturer s protool for Annexin V-FIT / PI stining (BD ApoAlert, BD Biosienes). Dt were nlyzed with ell Quest Softwre nd represent men7sem, nx1. Sttistil nlysis ws performed with ANOVA Two Ftor with Replition. Po.1 nd Po to 1.1% t nd 7 hours, respetively. Nnomolr onentrtions of did not indue poptosis t ny of the time points exmined (dt not shown). In HT kertinoytes, no signifint differenes in the perentges of ells in erly nd lte poptosis or nerosis were noted in ells treted with.1 mm t ll time points exmined. (1 mm) signifintly inresed the perentge of ells in erly nd lte poptosis t nd hours, respetively. Yet these inreses were very minor, with the totl perentge of HT ells in poptosis with eing o% of the ells (Figure, lte poptosis shown). In experiments with NHEK ells, no signifint differenes in the perentges of ells in erly or lte poptosis or nerosis were noted in ells treted with 13-is RA with the exeption of n inrese from 5.5 to.% in lte poptosis t hours in ells treted with 1 mm (Figure, lte poptosis shown). Apoptosis ws signifintly indued y sturosporine in SEB-1 seoytes, HT kertinoytes nd NHEK. No signifint differenes were noted etween stndrd medium nd ethnol ontrols in ny ell type t ny time point during these studies inditing tht the onentrtions of ethnol used in these experiments did not indue poptosis. speifilly inreses levels of leved spse 3 in SEB-1 seoytes SEB-1 seoytes were treted with nd four independent Western lots were run to detet leved spse 3. No leved spse 3 ws noted t hours in negtive ontrol lnes or in ells treted with. signifintly inresed leved spse 3 levels t nd 7 hours in SEB-1 seoytes (Figure 5). Speifilly,.1 mm nd 1 mm inresed expression of leved spse 3 on verge 3.5-fold (Po.1) nd fold (Po.1), respetively, t hours. Smll fold inreses were noted t 7 hours tht were not sttistilly signifint. Although the mgnitude of the inrese in leved spse 3 is gretest with 1 mm t hours, these results were not sttistilly signifint; due to the vriility indued y the very limited survivl of ells t this higher onentrtion. To determine if the indution of poptosis is speifi to, SEB-1 seoytes were lso treted with.1,.5, or 1 mm onentrtions of 9-is RA nd. Agin, no leved spse 3 ws deteted t hours in negtive ontrols or with ny onentrtion of either retinoid. Furthermore, unlike the se with, no signifint inreses in leved spse 3 were noted with either 9-is RA or t nd 7 hours (Figure 5 nd ). For dditionl onfirmtion tht the poptoti effet of 13- is RA is speifi to SEB-1 seoytes, Western lots for leved spse 3 were performed on NHEK. No leved spse 3 ould e deteted t ny time point exmined in NHEK ells treted with. However, leved spse 3 ws deteted when NHEK were treted with 1 mm sturosporine inditing tht these ells re ple of undergoing poptosis (Figure ). In summry, speifilly indues poptosis in dose-dependent mnner in 11

5 Reltive level of I. spse 3±SEM Reltive level I. spse 3±SEM Reltive level I. spse 3±SEM p19 p p19 p17 p19 p μm....5 μm P =.3..5 μm.5 μm 1 μm 9-is RA. P =..5 μm...5 μm Figure 5. inreses leved spse 3 protein in SEB-1 seoytes. () SEB-1 seoytes were treted with vehile,.1, 1, or 1 mm. ( nd ) Prllel experiments were performed with.1,.5, or 1 mm onentrtions of 9-is RA or. Blots were inuted with primry ntiodies to leved spse 3 (1:1,) nd tin (1:1,) for loding ontrol normliztion nd nlyzed y densitometry. p17 nd p19 re leved spse 3 frgments. Blots re representtive of minimum of four independent experiments. Grph represents normlized vlues reltive to vehile (ontrol) expression for four independent Western lots. Dt represent men7sem Po μm 1 μm SEB-1 seoytes ut not in NHEK. Furthermore, 9-is RA or does not indue poptosis in SEB-1 seoytes., ut not 9-is or, inreses TUNEL stining in SEB-1 seoytes To further test the hypothesis tht indues poptosis nd to onfirm the results from the Annexin V-FIT experiments in SEB-1 seoytes, we exmined the effets of on SEB-1 ells y TUNEL ssy. (.1 nd 1 mm) inresed the perentge of TUNEL-positive ells y 3.5- nd 5.7-fold, respetively (Pp.1) t hours, while eh onentrtion of inresed the perentge of TUNEL-positive ells y pproximtely 13-fold t 7 hours (Pp.1) (Figure 7). No differenes were noted t hours (dt not shown). To ompre the tions of to its isomeriztion produts, SEB-1 seoytes were lso treted with the sme onentrtions of 9-is RA nd nd no signifint inreses in TUNEL-stined ells were noted t ny time point exmined (Figure 7 nd ). Both 9-is RA nd hd 1 3% TUNEL-positive ells t time points exmined. Fenretinide tretment of SEB-1 seoytes signifintly inresed the perentge of TUNEL-positive ells in dose-dependent fshion t nd 7 hours (rnging from 15 to 5% positive ells) (Figure 7 nd ). No signifint inrese in the perentge of TUNEL-positive ells ws noted with retinoid X reeptor (RXR) pn gonist, D 35 t hours. However, 5 nm D 35 signifintly inresed TUNEL-positive ells from 3 to % t 7 hours (Po.1) (dt not shown). Apoptosis indution y in SEB-1 seoytes is not loked y RAR ntgonist AGN To determine if the effets of on poptosis re medited y RA reeptors, SEB-1 seoytes were treted with 1 mm in the presene of 1 mm AGN 19319, n RAR pn ntgonist, nd the TUNEL ssy ws performed. lone signifintly inresed the perentge of ells in poptosis over vehile ontrol y pproximtely 5-fold t nd 7 hours (Po.5). These inreses were not inhiited in the presene of AGN t nd 7 hours (Figure 7 nd d) (-hour dt not shown). To verify the tivity of AGN within our ells t the time points exmined in the TUNEL ssy, we performed quntittive PR for RAR responsive gene, tzrotene-indued gene 1 (TIG1). RAR tivtion indues the expression of TIG1 (Ngpl et l., 199). In the presene of 1 mm lone, TIG1 expression ws pproximtely 13- nd 17-fold higher thn ontrols t nd 7 hours, respetively. With the ddition of AGN 19319, TIG1 gene expression drmtilly dereses t nd 7 hours nd is lower thn vehile-treted ontrols (Figure 7e). is isomerized to over time in SEB-1 seoytes To study the kinetis of uptke in SEB-1 seoytes nd its possile isomeriztion to or 9-is RA, SEB-1 seoytes were treted with nd sujeted to HPL nlysis. remins reltively stle in stndrd ulture medium for pproximtely hours (Figure ). The 1 Journl of Investigtive Dermtology (), Volume 1

6 MM 5..5 μm onentrtion of in stndrd ulture medium lone is similr to the onentrtion in medium from SEB-1 seoyte-ontining pltes (Figure nd ). The onentrtions of within SEB-1 seoytes inreses to mximum of 35 ng/ml t 1 hours, t whih point the onentrtion delines for the durtion of the experiment (Figure ). The onentrtion of in the medium lone nd from pltes ontining SEB-1 seoytes ws muh lower thn onentrtions t the orresponding time points. The onentrtion of within SEB-1 seoytes egins to rise t 1 hours nd ontinues through the remining time periods. 9-is RA onentrtions re miniml t est in medium lone nd medium from SEB-1 pltes during the time ourse. Within SEB-1 seoytes, 9-is RA onentrtions rnge from 1. ng/ml t hour to mximum of 1 ng/ml t 7 hours, mgnitudes lower thn either 13-is RA or t the sme time periods (Figure ). DISUSSION Determining the tions of isotretinoin on the seeous glnd is essentil in dvning our understnding of the moleulr mehnism of tion of this drug nd in our serh for sfer therpeuti lterntives. Severl studies indite tht the effets of retinoids on ell prolifertion, ell yle, nd poptosis re retinoid speifi or ell-type speifi. For exmple, growth inhiition with hs een reported in humn rest ner ell lines, primry gliolstom ells, Epstein Brr virus-immortlized B lymphoytes, nd orl squmous ell rinom ell lines (Pomponi et l., 199; Ginnini et l., 1997; Tom et l., 1997; Bouterf et l., ). In some ses the effets noted with or 9-is RA were not duplited y (Bouterf et l., ). Most studies in other ell types suggest tht retinoids use lok in the G1/S phse of the ell yle triggering deresed S phse nd inresed the perentge of ells in G/G1 (Ginnini et l., 1997; Tom et l., 1997; rndll et l., ). It is lso well estlished tht retinoids indue poptosis in numerous ell types, oth norml ells nd tumor ell lines, lthough not previously demonstrted in seoytes. For exmple, in doses omprle to those given for the tretment of ne in humns, redues the survivl nd genesis of murine hippompl neurons in vivo (rndll et l., ; Ski et l., ). hs een shown to indue poptosis in primry nd metstti melnom ells (Zhng nd Rosdhl, ) s well s induing growth rrest followed y poptosis in oritl..5 μm s p19 p17 Figure. does not inrese leved spse 3 in NHEK. NHEK were treted with vehile,.1,.5, or 1 mm onentrtions of or 1 mm sturosporine (positive ontrol). Blots were inuted with primry ntiodies to leved spse 3 (1:1,) nd -tin (1:1,) for loding ontrol normliztion nd nlyzed y densitometry. Dt re representtive lot. firolsts from ptients with Grves disese (Psquli et l., 3). In OI/AML- retinoid-sensitive ell line sulones derived from leukemi ells, 9-is RA inhiited ell growth nd indued poptosis to greter extent thn or (Koistinen et l., ). These studies demonstrte tht the tions of retinoids re unique nd speifi to the model used. The ext mehnism of tion of in the tretment of ne remins lrgely unknown. hs little to no ility to ind to ellulr retinol-inding proteins or the RA nuler reeptors (RARs nd RXRs) (Levin et l., 199; Alleny et l., 1993; Fogh et l., 1993). It hs een suggested my, in ft, t s pro-drug tht is isomerized intrellulrly to, n gonist for RAR nuler reeptors, nd 9-is RA, whih is non-speifi gonist for oth RAR nd RXR nuler reeptors (Alleny et l., 1993; Ott et l., 199). Studies of immortlized humn seoyte line SZ95 showed tht is preferentilly metolized to, whih n ind to nd tivte RAR, whih leds to the overll inhiition of seoyte prolifertion (Tsukd et l., ). Our studies onfirm tht is primrily metolized to in SEB-1 seoytes eginning t hours. It is well estlished, however, tht is superior to either 9-is RA or for seosuppression (Geiger et l., 199; Hommel et l., 199; Ott et l., 199). Alterntively, my t in reeptor independent mnner y influening ellulr signling pthwys y diret protein intertions s demonstrted with other retinoids or y enzyme inhiition (Zorn nd Suro, 1995; Hoyos et l., ; Imm et l., 1; Krlsson et l., 3). Previous studies hve exmined the tions of, 9-is RA, nd on ultured humn seoytes, SZ95 immortlized seoytes, nd rt preputil ells (Zououlis et l., 1991, 1993; Tsukd et l., ; Wroel et l., 3). t onentrtions greter thn 1 7 M nd (1 5 to 1 M) signifintly deresed humn seoyte prolifertion fter 7 or 1 dys (Zououlis et l., 1991, 1993). Studies of immortlized humn seoytes SZ95, showed tht, 9-is RA, nd t onentrtions of 1 7 M signifintly redued prolifertion y pproximtely 5% fter 9 dys (Tsukd et l., ). In primry rt preputil ells, nd other RAR-seletive gonists signifintly deresed ell numers fter 9 dys (Kim et l., ). Proesses suh s ell yle rrest or poptosis my explin the histologil dt in humn skin iopsies tht demonstrtes drsti derese in the size, shpe, nd lipid ontent of seeous glnds fter 1 weeks of isotretinoin (Goldstein et l., 19) Sine prolifertion studies in SZ95 seoytes suggested tht the effets of nd other retinoids my e noted fter 7 9 dys, we designed experiments to look t the effets of, 9-is RA, nd on prolifertion, ell yle progression, nd poptosis in SEB-1 seoytes t time points lter thn hours ut prior to 7 dys. Our prolifertion studies show tht uses dose-dependent derese in ell ount fter nd 7 hours wheres 9-is RA nd show signifint dereses eginning t 7 hours. We would expet 13

7 9-is RA Fenretinide 1 μm AGN μm. 1 μm AGN Men±SEM % TUNEL positive μm Fenretinide d Men±SEM % TUNEL positive μm AGN 13RA 13RA+ 1 μm AGN e Reltive expression of TIG TiG1 gene expression + AGN Figure 7. The inrese in TUNEL stining with is not inhiited in the presene of RAR pn ntgonist AGN () Representtive imges of ontrol,.1, 1, nd 1 mm, 9-is RA,, nd fenretinide tretments t 7 hours ( hours dt not shown).() Quntifition of the perentge of TUNEL-positive-stined ells per tretment t nd 7 hours (9-is RA not shown). Dt represent men7sem, n ¼ 1. Sttistil nlyses were performed with ANOVA Two Ftor with Replition. Pp.1 nd Po.1. () Representtive imges of negtive ontrol, 1 mm, AGN 19319, nd 13-is RA omined with 1 AGN t 7 hours ( hours dt not shown). (d) Quntifition of the perentge of TUNEL-positive ells t 7 hours. Dt represent men7sem, n ¼ 1. Sttistil nlyses were performed with ANOVA Two Ftor with Replition. Pp.5 when ompred to ontrol; þ not sttistilly different. (e) Quntittive PR verifition of RAR ntgonist AGN tivity in SEB-1 seoytes. Brs represent the effiieny orreted normlized verge fold hnge of TIG1 under the experimentl onditions s determined y REST-XL softwre, n ¼. tht if our experiments were extended, the mgnitude of this derese would e greter s previously reported in SZ95 seoytes fter 9 dys (Tsukd et l., ). Overll, 13-is RA t the onentrtions tested in our study ts sooner in inhiiting prolifertion thn either 9-is RA or. These dt re supported y studies demonstrting n pproximte 3-fold derese in 3 H thymidine inorportion in SEB-1 seoytes tht were treted with for 7 hours. This derese is nerly -fold greter thn the dereses produed y 9-is RA or. This experiment suggests tht is more potent t growth inhiition thn either 9-is RA or in SEB-1 seoytes. Further supporting the hypothesis tht uses lok in the G1/S phse s demonstrted in other ell types, 1 Journl of Investigtive Dermtology (), Volume 1

8 ng/ml ng/ml ng/ml 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, Medium lone Hours Retinoids in SEB-1 medium Hours Retinoids in SEB-1 ells Hours 9-is RA 9-is RA 9-is RA Figure. is isomerized to within SEB-1 seoytes. HPL nlysis of () SEB-1medium lone, () medium removed from SEB-1 seoyte-ontining pltes, nd () SEB-1 seoytes fter 5 mm tretment for the indited times. Points re the verge of duplite smples. we show tht inreses p1 protein nd dereses ylin D1 protein t nd 7 hours. ylin D1 protein expression dereses y pproximtely 5% t 7 hours, whih oinides with our 3 H thymidine studies where 13-is RA hd the most striking effet t 7 hours. Furthermore, ylin D1 ws not deresed with 9-is RA or t 7 hours, whih is lso onsistent with our 3 H thymidine studies. No signifint inreses in p1 protein were noted with 9-is RA or lthough inresing trends were noted. Tken together, these experiment show tht in SEB-1 seoytes, is muh more effetive thn 9-is RA or in oth deresing the proportion of ells synthesizing DNA nd induing G1/S phse ell yle lok y inresing p1 nd deresing ylin D1 expression. Studies in SZ95 seoytes did not demonstrte poptosis in ells treted up to hours with (1 5 to 1 M) nd ssyed y DNA frgmenttion nd ltte dehydrogense relese (Wroel et l., 3). At hours, no hnges in poptosis were noted when SZ95 seoytes were treted with 1 7 M s ssessed y nnexin V stining, ell deth ssys, or FAS nlysis nd reverse trnsriptse-pr for the poptoti proteins, l- nd x. Interestingly, in SZ95 seoytes, inresed levels of spse 3 s deteted y FAS nlysis t hours. Aordingly, in our studies of SEB-1 seoytes, no inrese in poptosis ws noted hours fter tretment with s ssyed y Annexin V-FIT FAS. However, inreses in erly nd lte poptosis were noted t nd 7 hours with onentrtions of similr to those used in SZ95 seoytes, lthough the mgnitude of the numer of ells in poptosis is smll ompred to the positive ontrol, sturosporine. In ontrst, the mgnitude of the hnges in poptosis indued y ws muh greter in the TUNEL ssy. By extending the tretment times in our ssy we were le to detet the indution of poptosis y, whih ws verified y inresed expression of leved spse 3. Furthermore, the inrese in poptosis ws limited to s no signifint inreses in poptosis were noted when SEB-1 seoytes were treted with 9-is RA or. The effets of on poptosis nd growth inhiition my or my not e medited y retinoid reeptors. It is possile tht the effets of on poptosis nd growth inhiition my e medited y other isomeriztion produts suh s -oxo-isotretinoin or -hydroxy-isotretinoin (Orfnos nd Zououlis, 199). The -oxo metolites of retinoids hve een shown to e funtionlly tive in humn kertinoytes nd firolsts y their ility to indue hnges in gene expression (Bron et l., 5). Our dt show tht RAR pn ntgonist AGN suffiiently loks RAR tivtion in the presene of s mesured y signifint derese in TIG1 gene expression, yet does not lok poptosis indued y in SEB-1 seoytes, thus supporting the hypothesis tht poptosis indution y is independent of RAR tivtion. Alterntively, poptosis my e medited through RXR nuler reeptor tivtion (Zho et l., ). Using the RXR pn gonist D 35 (5 nm), signifint inrese in the perentge of TUNEL-positive SEB-1 seoytes ws noted t 7 hours. Although our HPL dt indite very low levels of 9-is RA ( mximum of 1 ng/ml t 7 hours), RXR tivtion y 9-is RA is possile (Alleny et l., 1993) or 13- is RA my e metolized to nother s yet unidentified metolite tht is ple of RXR tivtion. Alterntively, my hve effets tht re independent of retinoid reeptors. Interestingly, we showed tht fenretinide, retinoid known to indue poptosis primrily y RAR- nd RXR-independent mens is le to indue signifint poptosis in our SEB-1 seoytes. In ft, the degree of poptosis indued y fenretinide t hours is very similr to tht oserved with tretment t 7 hours. Fenretinide indues poptosis y elevting retive oxygen speies nd inreses in tivtion of ermide nd spses (Wu et l., 1). In ddition, retinoid-relted moleule, AGN indues poptosis without tivtion of the lssil retinoid reeptors (Keedwell et l., ; Blsurmnin et l., 5). It my e possile tht 13-is RA ts similrly to fenretinide or AGN vi 15

9 reeptor-independent mehnisms; lthough dditionl experiments re required to test this hypothesis. Sine the tions of retinoids differ in vrious ell types nd the effets of re most profound on seeous glnds in vivo, it is possile tht the indution of poptosis nd ell yle rrest my e speifi to seoytes sine 13-is RA filed to indue poptosis in HT kertinoytes or NHEK. It is possile tht with higher onentrtions of 13-is RA or longer tretment times tht poptosis my e indued in kertinoytes. Although there is no evidene in the literture of -indued poptosis in kertinoytes,, nd tzrotene, n RAR/g-seletive gonist, hve een shown to indue poptosis in HTs (Loufi et l., 3; Ppoutski et l., ). Tken together, these experiments support the hypothesis tht speifilly indues poptosis in SEB-1 seoytes nd not kertinoytes. In onlusion, our dt indite tht inhiits growth nd indues poptosis in SEB-1 seoytes nd not kertinoytes t onentrtions tht re therpeutilly hievle in humn plsm (Rollmn nd Vhlquist, 19; Admson, 199; Almond-Roesler et l., 199). Previous studies in humn seoytes nd immortlized seoytes hve lso doumented growth inhiition with, however, we hve extended these studies to show tht this growth inhiition is most likely due to influening the G1/S phse of the ell yle s evidened y deresed DNA synthesis, inresed p1 protein, nd deresed ylin D1 protein. In ddition, we report for the first time, tht lso indues poptosis in SEB-1 seeous ells. The ility of to indue poptosis is speifi to seoytes, not kertinoytes, nd is distint from effets oserved with 9-is RA nd tht my ount, in prt, for the superior effiy of in reduing seum prodution. Furthermore, the indution of poptosis y does not pper to involve RAR nuler reeptors. Eluidting the ellulr proesses tht re ffeted y in seoytes is step towrd understnding the overll moleulr mehnism of tion of this drug, whih my led to the identifition of lterntive strtegies for the tretment of ne. MATERIALS AND METHODS ell ulture The SEB-1 humn seoyte ell line ws generted y trnsfetion of seondry seoytes y SV Lrge T ntigen s previously desried (Thioutot et l., 3). SEB-1 ells were ultured nd mintined in stndrd ulture medium ontining: 5.5 mm low gluose DMEM 3:1 Hm s F1,.5% fetl ovine serum, hydroortisone. mg/ml, denine 1. 1 M, insulin 1 ng/ml, epiderml growth ftor 3 ng/ml, holer toxin M, nd ntiiotis. HT kertinoytes were ultured nd mintined in 5.5 mm low gluose DMEM, 5% fetl ovine serum, nd ntiiotis nd served s ontrol ell line in Annexin V-FIT FAS poptosis ssys. NHEK-neontl (pooled) (NHEK-neo, lonetis Kertinoyte System, mrex Biosiene, Wlkersville, MD) were ultured in kertinoyte growth medium- (mrex Biosiene, Wlkersville, MD). NHEK-neo ells served s ontrol ells in Annexin V-FIT FAS poptosis ssys nd Western lots for leved spse 3. Effets of retinoids on SEB-1 prolifertion Retinoid ompounds were purhsed through SIGMA (St Louis, MO): (R 355), 9-is RA (R 53) nd (R 5). Stok solutions of retinoids were hndled under dimmed yellow light, dissolved in 1% ethnol t onentrtion of 1 mm, nd stored under N gs t 1 until use. The RAR pn ntgonist AGN ws otined from Allergn (gift, Dr Rosh hndrrtn) nd dissolved in DMSO t onentrtion of 1 mm, nd stored t 71 until use. Tretments were mde from retinoid stoks diluted to the pproprite onentrtion in stndrd ulture mediums solutions under dimmed yellow light. Sturosporine (S 591, Sigm, St Louis, MO) ws soluilized in 1% ethnol t onentrtion of 1 mm, stored t 1, nd diluted to desired finl onentrtion in pproprite ell ulture medium for positive ontrol for poptosis. SEB-1 seoytes (pssges 3) were seeded t 1 ells per 35-mm plte nd grown until pproximtely % onfluent. Pltes were eh treted with.1,.5, 1 mm onentrtions of 13-is RA, 9-is RA,, or ethnol vehile lone (.1% or less) in triplite for,, nd 7 hours. ells were dethed using trypsin (.5%), olleted, nd diluted in stndrd ulture medium for mnul ell ounts using hemoytometer. ell viility ws ssessed using Trypn lue dye exlusion. Eh prolifertion ssy ws performed three independent times. Anlysis of vrine (ANOVA) Two Ftor with Replition ws used for nlysis. Results were onsidered signifint if Po.5. 3 H thymidine inorportion ssy SEB-1 seoytes (pssges 1 ) were t.5 1 ells per well in 1-well pltes nd grown until 3 % onfluent. Wells were rinsed with phosphte-uffered sline (PBS) prior to the ddition of.1,.5, or 1 mm onentrtions of, 9-is RA,, or ethnol vehile lone (.1% or less) in triplite wells in stndrd ulture medium. 3 H thymidine (1 mi/well) ws dded minimum of hours prior to the end of the tretment period. At the end of the tretment period, medium ws removed nd ells were rinsed twie with PBS, dethed using trypsin (.5%), nd olleted for liquid sintilltion ounting. Eh ssy ws performed minimum of three independent times. Sttistil signifine ws determined with ANOVA Two Ftor with Replition. Results were onsidered signifint if Po.5. Western lot nlysis for p1, ylin D1, nd leved spse 3 To onfirm results from ell prolifertion nd poptosis ssys, protein levels of p1, ylin D1, nd leved spse 3 were exmined using Western lot in our vrious ell lines. p1, ylindependent kinse inhiitor, loks progression through the G1/S phse of the ell yle. ylin D1 is speifilly required for progression into S phse. spse 3, the key exeutioner spse, is synthesized in the ell s pro-spse, whih then eomes leved nd tivted when ells undergo poptosis. Primry ntiodies for p1 Wf/ip1 (DS), ylin D1 (DS), leved spse 3 (Asp175), nd -tin nd s well s seondry nti-rit IgG horserdish peroxidse ntiody were otined from ell Signling Tehnology (Beverly, MA). primry ntiody nd 1 Journl of Investigtive Dermtology (), Volume 1

10 nti-mouse horserdish peroxidse-linked seondry ntiody were otined from Snt ruz Biotehnology IN (Snt ruz, A). SEB-1 seoytes (pssges ) were grown in 1-mm pltes in stndrd ulture medium until 5 75% onfluent. Pltes were rinsed with PBS nd then treted with: (.1, 1, nd 1 mm); 9-is RA (.1,.5, nd 1 mm); (.1,.5, nd 1 mm); ethnol vehile (.1% or less) s negtive ontrol; or 1 mm sturosporine dissolved in ethnol s positive ontrol. ells were treted for,, or 7 hours. NHEK ells (pssge 3) were grown in 1-mm pltes in stndrd ulture medium until pproximtely 5 75% onfluent. Pltes were rinsed with PBS nd then treted with: 13-is RA (.1,.5, nd 1 mm); ethnol vehile (.1% or less); or 1 mm sturosporine for,,, 1,,, or 7 hours. Totl ell protein lystes from dherent nd floting ells of SEB-1 seoytes, or NHEK were olleted, flsh frozen in liquid nitrogen, nd stored t 1 until needed. Protein onentrtion of eh smple ws determined y the BA Protein Assy (Piere, Rokford, IL). Equl mounts of protein were run on NuPge 1% or 1% Bis-Tris Gels with MES Running Buffer (Invitrogen Life Tehnologies, rlsd, A). Gels were trnsferred to polyvinylidene difluoride memrne, loked for 1 hour t room temperture in 5% non-ft dry milk, nd inuted with 1:1, dilution of leved spse 3 (Asp 175) (5A1) rit monolonl ntiody, 1:1, dilution of ylin D1, or 1:, 15, dilution of p1 Wf1/ip1 (DS) mouse monolonl ntiody. Seondry nti-rit IgG horserdish peroxidselinked ntiody nd nti-mouse horserdish peroxidse-linked ntiody were used to detet primry ntiodies. Supersignl West Pio hemiluminesent Sustrte (t no. 377, Piere, Rokford, IL) ws used for protein detetion. Blots were stripped with Restore Western Blot Stripping Buffer (Piere, Rokford IL) nd re-proed with -tin (no. 97 ell Signling Tehnologies) or tin (H3 t no. s1731) for loding ontrol. Films of lots were nlyzed nd quntified y densitometry with QuntityOne Softwre (Bio- Rd, Herules, A) fter kground sutrtion. Western lots were repeted minimum of three times. Dt ws nlyzed using Student s t-test nd results were onsidered signifint if Po.5. Annexin V-FIT/propidium iodide FAS poptosis ssy To determine if indues poptosis in SEB-1 seoytes nd the time ourse of this effet, the nnexin V-FIT FAS ssy ws hosen (Mrtin et l., 1995). Apoptosis ssys were performed in SEB-1 seoytes, HT kertinoytes, nd NHEK tht were treted with. SEB-1 seoytes (pssges ) nd HT kertinoytes (pssges 3 9) were seeded t 1 ells per 35-mm plte in their stndrd ulture mediums nd llowed to grow for 3 dys, feeding one efore tretment. Tretments onsisted of: stndrd ulture medium or ethnol vehile (.1% or less) s negtive ontrols, 1 mm sturosporine s positive ontrol, nd t finl onentrtions of.1 or 1 mm in SEB-1 seoytes nd HT kertinoytes for the initil studies. For follow-up studies exmining possile dose response, SEB-1 seoytes were sujeted to tretments of:.1, 1, 1 nm,.1, 1, nd 1 mm nd previously mentioned ontrols. All smples were run in triplite nd tretments were rried out for,,,,, nd 7 hours. In prllel experiments, NHEK ells (pssge 3) were grown in kertinoyte growth medium- until 7% onfluent. Tretments onsisted of ethnol vehile, 1 mm sturosporine, or (.1,.5, nd 1 mm). Smples were run in triplite nd ssyed t,,, 1,,, or 7 hours. Eh smple ws prepred ording to BD ApoAlert Annexin V Protool (t no. K5-1, BD Biosienes lonteh, Plo Alto, A). Ten thousnd events were olleted per smple using flow ytometry nd deris ws exluded y stter gting. Single Annexin V-FIT nd propidium iodide-stined smples s well s no-dye-negtive ontrol smples determined qudrnts for dt nlysis. Dt nlysis ws y ell Quest softwre (Beton Dikinson, nd) nd perentge of ells in erly poptosis, lte poptosis, nerosis, nd vile (unffeted) qudrnts were lulted nd ompred y ANOVA Two Ftor with Replition. Assy ws performed three independent times. Results were onsidered signifint if Po.5. TUNEL stining SEB-1 seoytes (pssges ) were ultured in 1-well pltes in stndrd medium until pproximtely 3 % onfluent. Wells were rinsed with PBS nd were treted in triplite with ethnol (.1% or less) s vehile ontrol,, 9-is RA, or eh in onentrtions of.1, 1, or 1 mm. Retinoids were diluted in stndrd ulture medium nd tretments were rried out for,, or 7 hours. In prllel experiments, SEB-1 seoytes (pssges ) were ultured in 1-well pltes in stndrd medium until pproximtely 3 % onfluent. Wells were rinsed with PBS nd were treted in triplite with: ethnol (.1% or less), DMSO (.1% or less), or ethnol nd DMSO together s vehile ontrols, 1 mm lone, 1 mm AGN lone, or 1 mm with 1 mm AGN Additionl experiments were performed with fenretinide, syntheti retinoid known to indue poptosis nd t vi retinoid reeptor-independent mehnism (Wu et l., 1). Fenretinide (-hydroxyphenyl-retinmide) ws hndled under dimmed yellow light nd dissolved in 1% ethnol to rete 1 mm stok solution stored t 1 (H 7779 Sigm, St Louis, MO). SEB-1 seoytes were treted in triplite with.1, 1, nd 1 mm onentrtions. Furthermore, experiments were performed with the RXR pn gonist D 35 (Glderm R&D, Sophi Antipolis, Frne). D 35 ws hndled under norml light onditions nd dissolved in DMSO to rete 1 mm stok solution stored t 1. SEB-1 seoytes were treted in triplite with 1 nd 5 nm onentrtions. ompounds were diluted in stndrd ulture medium nd pplied for or 7 hours. Eh well ws onsidered one smple. Smples were prepred y mnufturer s instrutions for in situ ell Deth Detetion Kit (Rohe Applied Siene, Indinpolis, IN). Additionl ssy ontrols inluded negtive ontrols of leling solution only nd DNse I-treted wells s positive ontrols. Results were nlyzed nd quntified y ounting positive stining ells/totl ells in three representtive fields per well for eh of the tretments rried out in triplite. Eh ssy ws performed three independent times; fenretinide nd D 35 experiments were repeted twie. Dt nlysis ws performed using ANOVA Two Ftor with Replition nd onsidered signifint if Po.5. Quntittive PR To verify RAR ntgonist tivity in TUNEL experiments, quntittive PR ws used to doument downregultion of the RAR trget gene, tzrotene-indued gene 1 (TIG1; retinoi id reeptor responder 1). SEB-1 seoytes were hndled, mintined, nd treted with nd RAR ntgonist AGN under experimentl 17

11 onditions tht were identil to those used in the TUNEL ssys. Totl RNA ws isolted nd quntittive PR performed s previously desried (Trivedi et l., ) Primer proe sets for TATA-inding protein (TBP; referene gene), nd retinoi id reeptor responder 1 (TIG1), were purhsed from Applied Biosystems (Foster ity, A) no templte nd no mplifition ontrols were inluded. The Reltive Expression Softwre Tool (REST-XL) ws used for dt nlysis. HPL is reported to isomerize minly to in other ell types inluding SZ95 seoytes (Tsukd et l., ). To eliminte the possiility of n lterntive pttern of isomeriztion nd to study the kinetis of uptke into SEB-1 seoytes, we utilized liquid liquid extrtion, reversed phse HPL with UV detetion. SEB-1 seoytes (pssge ) were grown to % onfluene in 1- mm pltes. For medium only ontrols, SEB-1 medium lone ws pled in 1-mm pltes. 5 mm ws pplied to SEB-1 seoytes nd medium only ontrol pltes in duplite for,,,, 1, 1,,, nd 7 hours. Experimentl smples inluded medium olleted from medium only ontrol pltes, medium from SEB-1 seoyte pltes, nd SEB-1 seoyte ell pellet. Smple preprtion ws y liquid liquid extrtion with ethyl ette. Ethyl ette ws evported nd the residue ws re-dissolved in mixture of etonitrile nd purified wter (/, vol/vol) efore injetion. Internl stndrd (itretin),, 9-is RA, nd stndrds nd qulity ontrols solutions were mde nd nlyzed to generte lirtion urve. Smples were injeted into Agilent 11 Series HPL system (Agilent Tehnologies, Plo Alto, A) using Nuleosil s (5 mm ) HPL olumns (Mherey-Ngel In., Düren, Germny). Smples were eluted in grdient solution omposed of purified wter nd etonitrile ontining.% eti id. Retinoid ompounds were deteted y UV detetion t 35 nm. ONFLIT OF INTEREST The uthors stte no onflit of interest. AKNOWLEDGMENTS We thnk Nte Sheffer of the ell Siene/Flow ytometry ore Fility of the Setion of Reserh Resoures, Penn Stte ollege of Mediine, for exellent tehnil ssistne with ll FAS experiments. We lso thnk Drs Johnnes Voegel nd Jen-lude ron of Glderm R&D for provision of ompounds nd ssistne with HPL nlysis, Dr Rosh hndrrtn nd Allergn In. for provision of ompounds, Anne Stnley of the Mromoleulr ore Fility of the Setion of Reserh Resoures, Penn Stte ollege of Mediine for ssistne with densitometry nlysis, nd helse Billingsley for providing tehnil ssistne. This work ws supported y NIH NIAMS R1AR AR7 to D.M.T. nd the Jke Gittlen ner Institute t the Pennsylvni Stte University ollege of Mediine. This work ws lso supported y NIH NIAMS R1AR AR7 to D.M.T. nd the Jke Gittlen ner Reserh Foundtion t the Pennsylvni Stte University ollege of Mediine. REFERENES Admson P (199) Phrmokinetis of ll-trns-retinoi id: linil implitions in ute promyeloyti leukemi. Semin Hemtol 31:1 7 Alleny G, Boquel MT, Sunders M, Kzmer S, Spek J, Rosenerger M et l. (1993) Retinoi id reeptors nd retinoid X reeptors: intertions with endogenous retinoi ids. Pro Ntl Ad Si USA 9:3 Almond-Roesler B, Blume-Peytvi U, Bisson S, Krhn M, Rohloff E, Orfnos E (199) Monitoring of isotretinoin therpy y mesuring the plsm levels of isotretinoin nd -oxo-isotretinoin. A useful tool for mngement of severe ne. Dermtology 19:17 1 Blsurmnin S, hndrrtn RA, Ekert RL (5) A novel retinoidrelted moleule inhiits pnreti ner ell prolifertion y retinoid reeptor independent mehnism vi suppression of ell yle regultory protein funtion nd indution of spse-ssoited poptosis. Onogene :57 7 Bldin V, Luks J, Mrote MJ, Pgno M, Drett G (1993) ylin D1 is nuler protein required for ell yle progression in G1. Genes Dev 7:1 1 Bron JM, Heise R, Blner WS, Neis M, Joussen S, Dreuw A et l. (5) Retinoi id nd its -Oxo metolites re funtionlly tive in humn skin ells in vitro. J Invest Dermtol 15:13 53 Bouterf H, Piht T, Kess D, Herold, Noll E, Blk PM et l. () Retinoids inhiit humn gliom ell prolifertion nd migrtion in primry ell ultures ut not in estlished ell lines. 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