Gender difference in the activity but not expression of estrogen receptors a and b in human lung adenocarcinoma cells

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1 Endocrine-Relted Cncer (26) Gender difference in the ctivity ut not expression of estrogen receptors nd in humn lung denocrcinom cells Susn M Dougherty 1, Willird Mzhwidz 1, Aimee R Bohn 1, Krist A Roinson 1, Kthleen A Mttingly 1, Kristy A Blnkenship 1, Mry O Huff 2, Willim G McGregor 3 nd Crolyn M Klinge 1 1 Deprtment of Biochemistry nd Moleculr Biology, University of Louisville School of Medicine, Louisville, KY 4292, USA 2 Deprtment of Biology, Bellrmine University, Louisville, KY 425, USA 3 Deprtment of Phrmcology nd Toxicology, Center for Genetics nd Moleculr Medicine, University of Louisville School of Medicine, Louisville, KY 4292, USA (Requests for offprints should e ddressed to C M Klinge; Emil: crolyn.klinge@louisville.edu) Astrct The higher frequency of lung denocrcinom in women smokers thn in men smokers suggests role for gender-dependent fctors in the etiology of lung cncer. We evluted estrogen receptor (ER) nd expression nd ctivity in humn lung denocrcinom cell lines nd norml lung firolsts. Full-length ER nd ER proteins were expressed in ll cell lines with higher ER thn ER. Although estrdiol (E 2 ) inding ws similr, E 2 stimulted prolifertion only in cells from femles, nd this response ws inhiited y nti-estrogens 4-hydroxytmoxifen () nd ICI 182,78. In contrst, E 2 did not stimulte repliction of lung denocrcinom cells from mles nd or ICI did not lock cell prolifertion. Similrly, trnscription of n estrogen response element-driven reporter gene ws stimulted y E 2 in lung denocrcinom cells from femles, ut not mles. Progesterone receptor (PR) expression ws incresed y E 2 in two out of five denocrcinom cell lines from femles, ut none from mles. E 2 decresed E-cdherin protein expression in some of the cell lines from femles, s it did in rest cncer cells, ut not in the cell lines from mles. Thus, ER nd ER expression does not correlte with the effect of ER lignds on cellulr ctivities in lung denocrcinom cells. On the other hnd, coctivtor DRIP25 expression ws higher in lung denocrcinom cells from femles versus mles nd higher in denocrcinom cells thn in norml humn ronchil epithelil cells. DRIP25 nd other ER coregultors my contriute to differences in estrogen responsiveness etween lung denocrcinom cells in femles nd mles. Endocrine-Relted Cncer (26) Introduction While the numer of women dying s result of metsttic rest nd colon cncer is declining, the mortlity ssocited with lung nd ronchus cncer in femles continues to rise (Greenlee et l. 2). Lung cncer is the leding cuse of cncer deth in oth women nd men in the United Sttes (Ptel et l. 24). Despite dvnces in chemotherpy for treting lung cncer, the 5-yer survivl rte hs not incresed significntly over the lst 25 yers, remining t pproximtely 14% (Willims & Sndler 21). The 2-fold higher frequency of lung cncer in women smokers thn in men smokers (Shields 2) strongly suggests the involvement of gender-dependent fctors in the etiology of lung cncer (Omoto et l. 21). Despite smoking for shorter periods of time, with fewer cigrettes per dy nd inhling less deeply thn men, women hve higher incidence of lung cncer, notly lung denocrcinom, type of non-smll cell lung cncer (NSCLC) (Stile & Siegfried 23). In ddition, women non-smokers re t 2.5-fold greter risk thn mle non-smokers for developing lung denocrcinom (Siegfried 21). Endocrine-Relted Cncer (26) DOI:1.1677/erc /6/ g 26 Society for Endocrinology Printed in Gret Britin Online version vi Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

2 S M Dougherty et l.: ER nd lung denocrcinom The mechnisms underlying the gender difference in NSCLC incidence is likely to e multifctoril. For exmple, women re more susceptile to smokinginduced DNA dmge thn men (Stile & Siegfried 23). However, muttions of p53 nd K-rs tht re regrded s erly events in crcinogenesis re rrely found in denocrcinoms, which ccount for % of lung cncer in femles (Hshimoto et l. 2). Three of the nine denocrcinom cell lines used in this study hve ctivting K-rs muttions, ut no genderdependent differences were pprent in this smll smple. Differences in the expression, gene polymorphisms nd ctivity of phse I (e.g. CYP1A1 (Mollerup et l. 1999)) nd II drug metolizing enzymes (e.g. glutthione-s-trnsferse (GST) nd N- cetyltrnsferse (NAT) (Stile & Siegfried 23)), my ply role in gender differences. Interestingly, over-expression of the protooncogene c-erb2/her2/ neu, lignd-independent epiderml growth fctor receptor, is ssocited with poor prognosis in NSCLC (Gtzemeier et l. 24) s well s rest cncer (Pegrm et l. 1998). The gender differences in denocrcinom implicte hormones in lung cncer risk. Estrogens increse the risk of rest cncer (Wolff et l. 1996) nd orl contrceptive therpy (OCT) is protective ginst ovrin nd endometril cncer (Boyle et l. 2). However, the role of estrogen in lung cncer is uncler. Some studies, reviewed y Stile nd Siegfried (23), indicte role for estrogen in lung cncer risk. For exmple, one study noted positive correltion etween post-menopusl estrogen replcement therpy, smoking nd lung denocrcinom (Tioli & Wynder 1994). A role for estrogen in the etiology of squmous cell crcinom (SCC) in Chinese popultion ws suggested y correltion etween SCC nd higher numer of menstrul cycles (Lio et l. 1996). Likewise, lrge clinicl tril in rest cncer ptients showed tht more women tking the ntiestrogen tmoxifen hd second primry non-rest cncer, including lung cncer, compred with those tking the romtse inhiitor exemestne, lthough the differences were not sttisticlly significnt (Coomes et l. 24). On the other hnd, the higher survivl rtes for women thn men with NSCLC in study of women my indicte protective effect of estrogen (Moore et l. 23). Indeed, recent study reported tht post-menopusl users of hormone replcement therpy (HRT) were t lower risk of developing lung cncer nd tht the protective effect of HRT ws minly oserved in current smokers who were lso the lightest smokers, i.e. <22 pckyers (Schth et l. 24). Clerly, further studies re needed to investigte the role of estrogens in lung cncer risk. Estrogens exert their moleculr ction y interction with two sutypes of estrogen receptor (ER), ER nd ER. In the originl chrcteriztion of ER nd ER mrna tissue distriution, in rts, ER ws predominnt in lung (Kuiper et l. 1997). There ws no reported lung phenotype for ER null (ERKO) mice (Couse et l. 1997, Runyi et l. 1997), ut comprison of the lungs of wild-type (wt) versus ER null (ERKO) mice reveled decresed numers of lveoli in dult femle ERx/x mice (Ptrone et l. 23). Lungs of femle ERKO mice lso showed decresed surfctnt, pltelet-derived growth fctor A (PDGF-A), nd grnulocyte-mcrophge colonystimulting fctor (GM-CSF), indicting tht ER plys role in lung homeostsis in mice (Ptrone et l. 23). Immunohistochemicl stining reveled ER expression in humn lung in columnr epithelium nd in intermedite, sl nd smooth muscle cells wheres ER ws expressed in sl nd smooth muscle cells (Tylor & Al-Azzwi 2). ER nd ER hve een proposed to ply opposite roles, i.e. yin nd yng, in cell prolifertion with ER eing prolifertive nd ER nti-prolifertive (Linderg et l. 23). For exmple, ER expression is incresed in rest cncer nd over-expression of ER inhiits estrdiol (E 2 )-stimulted rest cncer cell prolifertion (Pruthiyil et l. 24). Whether the ER :ER rtio increses in lung cncer is unknown. There re limited numer of studies with conflicting dt regrding ER nd ER expression in humn lung cncer nd in lung cncer cell lines (Croxtll et l. 1994, Cltgirone et l. 1997, Omoto et l. 21, Rdzikowsk et l. 22, Stile et l. 22, 25, Hersherger et l. 25) nd only two studies directly compring ER expression in lung cncer specimens nd cell lines in mles nd femles (Fsco et l. 22, Mollerup et l. 22). One study reported similr levels of ER nd ER in femles nd mles (Mollerup et l. 22). In contrst, nother group reported higher ER expression in femles thn in mles wheres ER expression ws similr (Fsco et l. 22). A generl conclusion from these reports is tht lung tumors from women re more likely to express ER thn tumors from men, with rnges from 7 97% y immunohistochemistry (IHC), nd tht the potentil role of estrogens in lung cncer is understudied (Ptel 25). It is well-estlished tht E 2 stimultes the prolifertion of estrogen-responsive tissues nd cell lines, e.g. humn rest nd endometril cells. In contrst to the mny inconsistent reports on ER expression, there re only four reports exmining the functionl Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

3 Endocrine-Relted Cncer (26) Tle 1 Chrcteristics of the humn non-smll cell lung cncer (NSCLC) cell lines used in this report. All cell lines were otined from ATCC Mnss, VA, USA Cell line Sex of ptient Smoker/nonsmoker (NS) Reported ER sttus (reference) K-rs muttion (codon GGT-GTT; G12C) ( unless otherwise indicted) mle unknown no ER (Croxtll et l., 1994) Low ER nd high ER (Stile et l., 22) No ER, ut ER (Hersherger et l., 25) NCI- mle smoker Low ER nd high ER (Stile et l., 22) ER nd ER (Pietrs et l., 25) K-rs point muttion (Okudel et l., 24) K-rs point muttion (ATCC informtion) NCI- mle smoker No K-rs muttion, ut mutnt N-rs NCI-H1792 mle smoker K-rs point muttion NCI-H1395 femle smoker No K-rs muttion NCI-H1435 femle NS ER > ER (Mollerup et l., 22) No K-rs muttion ER; < 1% of the level of ER; in cells (Mollerup et l., 22) NCI-H1793 femle NS No K-rs muttion NCI- femle smoker K-rs muttion NCI-H273 femle smoker No K-rs muttion consequences of ER expression in lung denocrcinom cell lines (Stile et l. 22, 25, Hersherger et l. 25, Pietrs et l. 25). The studies reveled tht E 2 stimulted prolifertion of lung denocrcinom cell lines from mles, e.g. NCI- (Tle 1), in vitro in n ER-dependent mnner (Stile et l. 22, 25, Pietrs et l. 25) nd one study reported tht E 2 incresed E-cdherin nd Id-2 expression in 21T cells, lso from mles (Hersherger et l. 25). The vriility of ER nd ER expression etween studies nd the pucity of functionl studies on ER in lung denocrcinom indicte tht more informtion is needed to determine the significnce nd role of these receptors in NSCLC. In prticulr, no one hs directly compred the effect of E 2 nd nti-estrogens on the prolifertion or estrogenic responses of lung denocrcinom cell lines from mle versus femles in side-y-side comprison. In this study, we used nine different humn denocrcinom cell lines from mle or femle lung cncer ptients to test their estrogen responsiveness. We demonstrte tht the humn denocrcinom cell lines, norml humn lung firolst cell line (NF164), nd norml humn ronchil epithelil (NHBE) cells express ER nd ER proteins. Although no differences in the levels of ER nd ER proteins or in [ 3 H]E 2 inding in cells from mles or femles were detected, there were significnt differences in cellulr iochemicl responses to E 2 nd the nti-estrogens 4-hydroxytmoxifen () nd ICI 182,78 etween cell lines from mles nd femles. Wheres the prolifertion of the cell lines from femles ws stimulted y E 2 nd locked y concomitnt dministrtion of or ICI 182,78, the cell lines from mles were non-responsive to these tretments. Similr results were detected t the trnscriptionl level. Although other investigtors hve demonstrted responses of lung denocrcinom cell lines from mles to selective ER modultors (SERMs) including fulvestrnt (Stile et l. 22, 25, Hersherger et l. 25), our dt support the possile use of SERMs such s tmoxifen or fulvestrnt (ICI 182,78) for selectively treting women with lung denocrcinoms, lthough further studies will e needed to extend these suggestions. Mterils nd methods Chemicls E 2 nd 4-hydroxytmoxifen () were purchsed from Sigm. ICI 182,78, 4,4,4 -(4-propyl-[1H]- pyrzole-1,3,5-triyl)trisphenol (PPT, n ER-selective gonist) nd 2,3-is(4-hydroxyphenyl)-propionitrile (DPN, n ER-selective gonist) were purchsed from Tocris (Ellisville, MO, USA). The R,R enntiomer of 5,11-cis-diethyl-5,6,11,12-tetrhydrochrysene-2,8-diol (R,R-THC) ws generously provided y Dr J A Ktzenellenogen of the University of Illinois (Sun et l. 1999) Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

4 S M Dougherty et l.: ER nd lung denocrcinom Cell lines, NCI-, NCI-, NCI-H1395, NCI- H1435, NCI-H1792, NCI-H1793, NCI-, NCI- H273 nd cell lines were purchsed from ATCC (Mnss, VA, USA) nd were mintined in the recommended medi nd supplements. The chrcteristics of the lung denocrcinom cell lines re listed in Tle 1. The primry firolst cell strin GM164 (Coriell Institute, Cmden, NJ, USA) ws originlly derived from humn fetl lung tissue, nd ws immortlized y expression of the ctlytic suunit of humn telomerse (htert), under license from Geron Corportion (Menlo Prk, CA, USA), y Dr L McDniels (University of Texs Southwestern Medicl Center, Dlls, TX, USA) nd nmed NF164. The cells were kryotyped nd found to e 46XY (Ouellette et l. 2). They were provided to W G M y Dr McDniels under the terms of Mteril Trnsfer Agreement 325 etween W G M nd Geron Corportion. NHBE cells were purchsed from Cmrex (Wlkersville, MD, USA) nd mintined in ronchil epithelil growth medium supplemented with BulletKit growth fctors (Cmrex). Cell prolifertion/mtt ssy Cell prolifertion ws determined using the Cell Prolifertion Kit 1 (MTT) (Roche) nd Cell Titer 96 AQueous One solution cell prolifertion ssys (Promeg) ccording to the mnufcturers protocols. Briefly, 2r1 3 cells were plted per well in 96-well pltes in phenol-red-free medi contining 1% dextrn-coted chrcol-stripped fetl ovine serum (DCC-FBS) for 24 h prior to tretment. The cells were treted with ethnol (), ICI 182,78, E 2, trns-resvertrol, or other compounds (see figure legends for detils) for 4 dys. Tretments were replenished fter 48 h. The sornce of soluilized formzn product ws mesured t 57 nm (MTT) nd 49 nm (Cell Titer 96) directly in ech well. Within ech experiment, ech tretment ws performed in qudruplicte nd vlues were verged. Vlues were compred with those in the wells treted with vehicle () control which ws set to 1%. At lest four seprte experiments were performed for ech cell line. RNA extrction nd quntittive rel time RT-PCR Cells were grown to 7% confluency in 1 mmr2 mm cell culture dishes in phenol-red-free medi contining 1% DCC-FBS for 24 h prior to tretment with (vehicle control), E 2 or other compounds indicted in the text for 6 h. Cells were wshed three times in PBS, nd RNA ws extrcted using the TRIzol regent (Invitrogen) ccording to the mnufcturer s protocol followed y purifiction on RNesy columns (Qigen, Vlenci, CA, USA). RNA qulity ws determined using the RNA 6 Nno Assy kit on the Agilent 21 Bionlyzer (Wilmington, DE, USA). RNA concentrtion ws determined y sornce t 26 nm. Totl RNA ws reverse-trnscried using rndom hexmers nd the High Cpcity cdna rchive kit (PE Applied Biosystems (ABI), Foster City, CA, USA). The QIAquick PCR purifiction kit (Qigen) ws used to purify cdna. ER, ER nd 18S rrna Tqmn primers nd proes were purchsed s Assys-on-Demnd Gene Expression Products (ABI). Expression of genes ws determined for ech trget gene in triplicte using 2 ng of purified smple cdna in ech experiment nd replicted twice for totl n=3. Ech smple ws normlized using 18S rrna. The efficiency of ech primer/proe set ws evluted y concentrtion optimiztion of primers, proes nd input cdna generting stndrd curve with seril dilutions of untreted RNA. The efficiencies of rel-time PCR of ER, ER nd 18S were 99.5, 99.5 nd 97.9% with correltions of.986,.968 nd.942 respectively. Rel-time PCR ws performed in the ABI PRISM 77 SDS 2.1 using reltive quntifiction with the following therml cycler conditions: hold t C for 2 min nd 95 C for 1 min, followed y 4 cycles of 95 C for 15 s nd 6 C for 1 min in 2 ml rection in 96-well pltes. Ech 96-well plte lso included minus RT nd rection mix without cdna s negtive controls. Anlysis nd fold differences were determined using the comprtive cycle threshold (C T ) method s descried in ABI Technicl Bulletin 2 (Bustin 22, Ginzinger 22). Fold chnge ws clculted from the DDC T vlues with the formul 2 xddc T nd is given s percentge reltive expression compred with cells. Preprtion of whole-cell extrcts (WCEs) Ech cell line ws grown in its corresponding culture medium supplemented with 1% DCC-FBS, 1% penicillin/streptomycin plus the indicted concentrtions of chemicls for 24 h prior to hrvest. When cell tretment experiments were performed, cells were grown in phenol-red-free medi supplemented s ove plus the tretment(s) indicted in the figure legends nd text. WCEs were prepred in RIPA uffer ( mm Tris HCl, ph 7.4; 1% Nonidet P-4;.25% Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

5 Endocrine-Relted Cncer (26) N-deoxycholte; 1 mm NCl; 1 mm EDTA; 1 mm phenylmethylsulfonyl fluoride (PMSF); protinin, leupeptin nd pepsttin, ech t 1 mg/ml; 1 mm N 3 VO 4 ; 1 mm NF). Protein concentrtions were determined using BioRd s DCC ssy (Hercules, CA, USA). Specific [ 3 H]E 2 inding ssy Nucler nd cytoplsmic frctions (NE nd CE) were prepred from ech cell line using the NE-PER nucler nd cytoplsmic extrction regents (Pierce, Rockford, IL, USA) ccording to the mnufcturer s recommendtions. Specific [ 3 H]E 2 inding to culovirusexpressed recominnt humn ER (rher) nd rher (Kulkosky et l. 22, Kulkosky & Klinge 23) or to the NE, CE nd WCE from the lung cell lines ws mesured in the presence of 2-fold molr excess of cold E 2 y dsorption to hydroxyptite (HAP) s previously descried (Kulkosky et l. 22). Western lotting Identicl mounts of whole-cell lyste extrct (25 4 mg protein) were seprted on 1% SDS-PAGE gels nd electrolotted on to polyvinylidene fluoride (PVDF) memrnes (Pll Corportion, Penscol, FL, USA). As positive nd negtive controls, known mounts (in fmoles from HAP ssys) of culovirus-expressed rher nd rher, purchsed from Pnver or produced in Sf21 cells (Kulkosky et l. 22, Kulkosky & Klinge 23) were seprted in prllel with WCE smples. The trnsfer ws monitored y pre-stined moleculr weight mrkers (Precision Stndrd, BioRd). Following trnsfer, memrnes were locked in 5% milk Trisuffered sline (TBS) Tween then proed with ntiodies: nti-er monoclonl ntiody AER32 recognizes (LVision-Neomrkers, Fremont, CA, USA); nti-er polyclonl ntiody H-1 (Snt Cruz Biotechnology, Snt Cruz, CA, USA) recognizes 1 1 of her nd N-19 (Snt Cruz Biotechnology) lso recognizes the N-terminus of her; nti-progesterone receptor (PR) monoclonl ntiody AB-8 recognizes the N-terminl hlf of hpr (LVision-Neomrkers). E-cdherin ntiody (no. 465) ws purchsed from Cell Signling (Beverly, MA, USA). Blots were stripped nd re-proed with nti--ctin (Sigm) or -tuulin (LVision-Neomrkers) to normlize for protein loding. The ntiody to DRIP25 ws kindly provided y Dr M Gredin (Pined Torr et l. 24). Super Signl West Pico Chemiluminescent Sustrte (Pierce) ws used to detect protein nds. Resulting immunolots were scnned into Adoe Photoshop 7. using Microtek ScnMker III scnner (Crson, CA, USA). Un-Scn-It (Silk Scientific, Orem, UT, USA) ws used to quntitte the integrted opticl densities (IOD) for ech nd. The IOD for ER, ER, PR, nd E-cdherin were divided y concordnt -ctin IOD in the sme lot. Trnsient trnsfection ssy For trnsient trnsfection, cells were plted in 24-well pltes t density of 1.5r1 4 cells/well in phenol-redfree OPTI-MEM I reduced serum medium (Gico/ Invitrogen) supplemented with 1% DCC-FBS, 1% penicillin/streptomycin. Trnsient trnsfection ws performed using FuGene6 (Roche). Ech well received 2 ng of pgl3-pro-luciferse reporter (Promeg) contining two tndem copies of consensus estrogen response element (ERE; i.e. EREc38 (Tyulmenkov et l. 2)) nd 5 ng Renill luciferse reporter (prl-tk) from Promeg. At 24 h fter trnsfection, triplicte wells were treted with (vehicle control), E 2,, DPN, PPT, ICI 182,78, resvertrol or other compounds s indicted in the figures. The cells were hrvested 3 h post-tretment using Promeg s Pssive Lysis uffer. Luciferse nd Renill luciferse ctivities were determined using Promeg s dul-luciferse ssy in Plte Chmeleon luminometer (BioScn, Wshington, DC, USA). Firefly luciferse ws normlized y Renill luciferse to correct for trnsfection efficiency. Fold induction ws determined y dividing the verged normlized vlues from ech tretment y the vlue for ech trnsfection condition within tht experiment. Vlues were verged from multiple experiments s indicted in the figure legends. Sttistics Sttisticl nlyses were performed using Student s t test or one-wy ANOVA followed y Student Newmn Keuls or Dunnett s post-hoc tests using GrphPd Prism (Sn Diego, CA). Results E 2 stimultes, nd nd ICI 182,78 inhiit, the prolifertion of femle lung denocrcinom cell lines. We exmined the effect of E 2,, ICI 182,78 nd R,R-THC ( selective ER gonist/er ntgonist (Sun et l. 1999)) lone or in comintion on the prolifertion of lung denocrcinom cell lines from femles (Fig. 1) nd mles (Fig. 2). Of the cell lines from femles, H1395 showed the most stimultion y E 2 nd H273 showed the lest. The mgnitude of Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

6 S M Dougherty et l.: ER nd lung denocrcinom Reltive prolifertion E 2 (nm) 1 µm, A + + ICI + R,R-THC Reltive prolifertion 1 H E 2 (nm) 1 µm, B + + ICI + R,R-THC Reltive prolifertion H E 2 (nm) 1 µm,, + + ICI C + R,R-THC Reltive prolifertion H E 2 (nm) 1 µm, + + ICI D 1 µm R,R-THC + R,R-THC Reltive prolifertion H E 2 (nm) 1 µm + + ICI 1 µm R,R-THC E + R,R-THC Figure 1 E 2 stimultes nd nti-estrogens inhiit the prolifertion of lung denocrcinom cell lines from femles. (A), H1793 (B), H1395 (C), H1435 (D) nd H273 (E) lung denocrcinom cells, ll from femles, nd humn rest cncer cells (F) were treted with the indicted concentrtions of E 2,, ICI 182,78 nd R,R-THC; or the indicted comintions thereof for 4 dys with medi (plus tretment) chnged on dy 2. Cell prolifertion ws mesured y MTT ssy. Ech r represents the ments.e.m. of t lest six independent experiments., significntly different from control () vlues;, significntly different from the lone vlue, P<.5. E 2 -induced prolifertion is consistent with tht seen in rest cncer cells (Fig. 1F) nd reported for the 21T denocrcinom cell line (Hersherger et l. 25). E 2 stimultion ws locked y which is n ctive metolite of the drug tmoxifen, used cliniclly to lock ER ction in rest cncer (Jordn & Ppps 23) nd ICI 182,78 (Fslodex), pure steroidl ER ntgonist. inhiited prolifertion in ll cell lines from femles. The inhiition of E 2 - induced prolifertion y R,R-THC, n ER-selective ntgonist/er gonist (Sun et l. 1999), ws similr to tht with ICI 182,78, ut less thn, suggesting the possiility tht ER my hve role in cell repliction. In contrst to the results in the lung denocrcinom cell lines from femles, E 2 hd no effect on the prolifertion of NF164 (Fig. 2A) or ny of the denocrcinom cells from mles (Fig. 2B E). The lck of E 2 -induced prolifertion of cells contrdicts the stimultory effect of E 2 reported previously Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

7 Endocrine-Relted Cncer (26) Reltive prolifertion NF E 2 (nm) 1 µm 182,78 + ICI + + R,R-THC A 1 µm R,R-THC Reltive prolifertion E 2 (nm) 1 µm 182,78 + ICI + B + R,R-THC Reltive prolifertion E 2 (nm) C E 2 (nm) 1 µm 182,78 + ICI + + R,R THC 1 µm R,R-THC Reltive prolifertion 125 H µm 182, ICI D 1 µm R,R-THC + R,R-THC Reltive prolifertion E 2 (nm) 1 µm 182,78 + ICI + E + R,R-THC Figure 2 ER lignds do not ffect the prolifertion of NF164 norml lung firolsts or denocrcinom cell lines from mles. NF164 (A), (B), (C), H1792 (D) nd (E) lung denocrcinom cells, ll from mles, were treted with the indicted concentrtions of E 2,, ICI 182,78 nd R,R-THC; or the indicted comintions thereof for 4 dys s descried in the Mterils nd methods section nd Fig. 1. Cell prolifertion ws mesured y MTT ssy s descried in the Mterils nd methods. Vlues re expressed s reltive to control. Ech r represents the ments.e.m. of t lest six independent experiments;, significntly different from control () vlues, P<.5. (Stile et l. 22). The reson for this discrepncy my result from the 24-h serum-free incution of the cells prior to E 2 tretment in the previous report (Stile et l. 22). In contrst, we mintined ll cell lines in 1% DCC-FBS for 24 h prior to hormone tretment. Thus, the hormone/lignd effects we mesured my e reduced in mgnitude compred with serum-deprived cells. ICI 182,78 (Fslodex) hd no effect on the prolifertion of lung denocrcinom cell lines from mles. inhiited the prolifertion of nd H1792 cells y 2% (Fig. 2C nd D). In the cse of, the comintion of E 2 nd lso inhiited prolifertion y pproximtely 25%. Notly, lone or comined with E 2 hd greter inhiitory effect on the prolifertion of denocrcinom cells from femles (Fig. 1) thn mles; i.e. reducing prolifertion y % versus 2% respectively. R,R-THC did not ffect the prolifertion of ny of the cell lines from mles, whether lone or in comintion with E 2. We conclude tht lung denocrcinom cell lines from femles re more responsive to E 2,, ICI 182,78 nd R,R-THC compred with cell lines from mles Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

8 S M Dougherty et l.: ER nd lung denocrcinom C T / C T smple H1395 H1435 H1792 H1793 A ERα ERβ H273 NF164 NHBE AER32 ERα β-ctin AER32 ERα β-ctin NF164 H1792 NHBE NHBE ERβ ntiody H1 ERβ1 H1435 NHBE NF164 H273 H1435 H1792 H1793 NF164 rherα rherα NF1793 H273 C kd kd 1 37 ERβ1 β-ctin H1395 NHBE H1792 H1793 H1435 H1435 H1395 H1395 NF164 ERβ ntiody N-19 rherβ1 NF164 rherα H1792 NF164 E kd B kd fmoles rherβ D β-ctin ER (fmol/µg WCE) ERα ERβ AVG. fmol/µg mle ERα femle ERβ F 2 H1395 H1435 H1792 H1793 H273 NF164 NHBE 12 Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

9 Endocrine-Relted Cncer (26) Specific [ 3 H]E 2 inding (moles/µg protein) 3.5E-16 3.E E-16 2.E E-16 1.E-16 5.E-17 * G Specific [ 3 H]E 2 Binding (moles/µg) 3.5E-16 3.E E-16 2.E E-16 1.E-16 5.E-17 mle femle H.E+.E+ mle femle Nucler Cytosolic Figure 3 ER nd ER re expressed in lung denocrcinom cells. (A) Quntittion of mrna expression of ER nd ER y rel-time RT-PCR ws performed s descried in the Mterils nd methods section. Vlues re the mensts.e.m. of qudruplicte determintions nd re normlized y ER nd ER expression in cells. (B nd C) Western lots for ER using ER ntiody AER32. (D nd E) Western lots for ER using ER ntiodies N-19 nd H-1 respectively. The lots were stripped nd re-proed for -ctin expression for normliztion. Known mounts of rher (25 nd 62 fmol) nd rher (1, 225 nd 3 fmol), determined y HAP ssy s descried in the Mterils nd methods, were seprted in prllel with the lung smple WCE. Estimtion of ER nd ER1 concentrtions ws performed s descried in the Mterils nd methods. (F) The expression of ER nd ER1 from different WCE preprtions ws quntitted y Western lotting. Ech r represents the ments.e.m. of t lest five independent experiments. The inset shows the ments.e.m. for vlues from the denocrcinom cell lines from mles nd femles;, significntly different from ER expression in, P<.1;, significntly different from ER1 expression in ny other cell line, P<.1; s determined y one-wy ANOVA followed y Student Newmn Keuls multiple comprison test. Specific [ 3 H]E 2 inding to WCE (G) nd CEs nd NEs (H) ws mesured y HAP ssy s descried in the Mterils nd methods. Vlues re the mensts.e.m. of triplicte determintions. *Sttisticlly different from vlues for lung denocrcinom cell lines from mle nd femle, P<.1; one-wy ANOVA followed y Student Newmn Keuls multiple comprison test. Expression of ER nd ER mrna nd protein in lung denocrcinom cells One possile explntion for the lck of estrogen responsiveness in the lung denocrcinom cells from mles is tht they do not express ER or express ER t reduced levels compred with the cells from femles. Of the nine denocrcinom cell lines used in this study, the ER sttus hs een reported in, nd H1435 (Tle 1). It is noteworthy tht contrdictory findings were reported (Tle 1). Quntittive rel-time RT-PCR ws used to determine ER nd ER mrna expression in NHBE, NF164 nd the lung denocrcinom cell lines (Fig. 3A). Fold differences in gene expression were determined y the comprtive C T method using 18S rrna s the endogenous control. cells were used s the clirtor nd expressed more ER mrna thn NHBE, NF164 or ny of the lung denocrcinom cell lines. ER mrna expression ws higher thn ER expression in ll the lung cell lines. There ws no difference in ER mrna expression etween NHBE, NF164 nd the lung denocrcinom cell lines.,, H1395, H1792, H1793, H273 nd NF164 expressed higher levels of ER mrna thn cells. To determine ER nd ER protein expression, known concentrtions, s ssyed y [ 3 H]E 2 inding (Kulkosky et l. 22), of culovirus-expressed rher nd rher protein were seprted in prllel with the WCEs of the lung denocrcinom cell lines nd used s clirtors to estimte ER nd ER expression levels y Western lot (Fig. 3B E). All cell lines expressed full-length ER nd no shorter ER products were detected (Fig. 3B nd C). These dt indicte tht intct ER is expressed in the denocrcinom cell lines. Our dt differ from report tht, nd expressed ER s 8 nd 54 kd nds, ut tht only expressed 67 kd ER (Stile et l. 22). ER expression in NHBE nd ll denocrcinom cell lines ws similr to cells wheres NF164 ws lower (Fig. 3F). As ntiodies hve history of showing crossrectivity with ER, we used two different ntiodies to exmine ER expression. We hve demonstrted the specificity of the AER32 ER nd H1 ER ntiodies for their respective ER sutype (Klinge et l. 25). The N-19 ntiody only recognized the Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

10 S M Dougherty et l.: ER nd lung denocrcinom Fold induction 1.6 NF164 A nm E 2 1 nm & 182,78 & ICI Fold induction nm E 2 1 nm & 182,78 B & ICI Fold induction nm E 2 1 nm & 182,78 C & ICI Fold induction nm E 2 1 nm & 182,78 & ICI D Fold induction H nm E 2 1 nm & 182,78 & ICI E Figure 4 Lck of estrogen responsiveness of NF164 nd mle lung denocrcinom cells in trnsient trnsfection ssy. NF164 (A), (B), (C), (D) nd H1792 (E) mle denocrcinom cells were trnsiently trnsfected with n ERE-luciferse reporter s descried in the Mterils nd methods section. The cells were treted with the indicted concentrtions of E 2, 1 nm nd 1 mm ICI 182,78, lone or in comintion s indicted for 24 h. Luciferse nd Renill luciferse were ssyed s descried in the Mterils nd methods. Luciferse vlues were normlized y Renill luciferse vlues nd the reltive light unit (RLU) vlue for (vehicle control) ws set to 1. Vlues re the mensts.e.m. of three seprte trnsient trnsfection ssys;, significntly different from control (P<.5, Student s t test). ER1 6 kd nd (Fig. 3D), i.e. the long isoform, nd not the ER1s short isoform of ER (Scoie et l. 22). Western lots using the H1 ER ntiody indicted tht most cells express the ER1 nd ER1s isoforms s nd 53 kd sizes respectively (Fig. 3E). A summry of the estimted ER nd ER expression in the cells sed on Western lotting is presented in Fig. 3F. ER expression ws greter thn ER expression in ll lung denocrcinom cell lines, nd in NHBE nd NF164 cells thn in cells. NHBE cells showed higher ER expression thn NF164, or ny of the lung denocrcinom cell lines. Others hve reported ER1 migrting s kd in humn tissues (Choi et l. 21) nd ER from mouse lung ws 65 kd (Ptrone et l. 23). As ER splice vrints ER2/cxL nd ER2/cxS re expressed in humn tissues s kd proteins, it is possile tht the lower ER nd detected in the Western lots shown here my represent more thn one isoform of ER. Further testing will e Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

11 Endocrine-Relted Cncer (26) Fold induction H A Fold induction H B. 1 1 nm E 2 1 nm & 182,78 & ICI. 1 1 nm E 2 1 nm & 182,78 & ICI Fold induction H1793 C Fold induction D. 1 1 nm E 2 1 nm & 182,78 & ICI nm E 2 1 nm & 182,78 & ICI Fold induction H nm E 2 1 nm & E 182,78 & ICI Fold induction nm E 2 1 nm & 182,78 F & ICI Figure 5 E 2 stimultes endogenous ER trnscriptionl ctivity in femle humn lung denocrcinom cells s determined y trnsient trnsfection ssy. H1395 (A), H1435 (B), H1793 (C), (D) nd H273 (E) femle denocrcinom cells nd rest cncer cells (F) were trnsiently trnsfected with n ERE-luciferse reporter; they were then treted, hrvested nd ssyed s descried in the Mterils nd methods section, nd Fig. 4. Note tht these cells were not trnsfected with ER., Significntly different from control;, significntly different from the lone vlue (P<.5, Student s t test). required to exmine this possiility. There ws no correltion etween the expression of the shorter ER protein nd estrogen responsiveness or gender from which the cells were otined. These dt indicte tht it is unlikely tht over-expression of the dominnt negtive ER2/cx isoform ccounts for differences in estrogen responsiveness etween cells. [ 3 H]E 2 inding to endogenous ER in lung denocrcinom cells Another explntion for the lck of E 2 response in the denocrcinom cells from mles is tht the expressed ER is non-functionl. One functionl mesure of ER expression is specific [ 3 H]E 2 inding Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

12 S M Dougherty et l.: ER nd lung denocrcinom PR-B PR-A kd A β-ctin PR-B β-ctin H1395 H1435 H273 NHBE H kd PR-B PR-A β-ctin NF164 H1793 H kd control B 3 Reltive PR NHBE NF164 H1395 H1435 H1792 H1793 H273 E-cd β-ctin NF kd -1 E-cd -1 β-ctin H kd -1 C -1 - E-cd β-ctin H1792 H1793 NF kd H273 H1435 NHBE E-cd - β-ctin kd Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

13 Endocrine-Relted Cncer (26) control D 3 Reltive E-cdherin/β-ctin H1395 H1435 H1792 H1793 H273 NHBE NF164 Figure 6 PR is mrker of estrogen responsiveness in some of the lung denocrcinom cell lines from femles nd E-cdherin is not mrker of estrogen response. Cells were treted with (vehicle) or for 24 h. WCEs (35 mg) were seprted on 1% SDS-PAGE gels, trnsferred to PVDF memrnes nd immunolotted for PR (A). PR-B is 116 kd nd PR-A is 81 kd. Memrnes were stripped nd re-proed for -ctin s loding control. (B) MentS.E.M. (n=3 5) of the totl PR (PR-B+PR-A) normlized y -ctin nd with -treted PR expression set to 1%;, significntly different from control (P<.1). (C) Indicted cell lines were treted with (vehicle) or for 24 h. WCEs (35 mg) were seprted on 12% SDS-PAGE gels, trnsferred to PVDF memrnes nd immunolotted for E-cdherin. Moleculr weight mrkers run in prllel in ech gel (indicted in kd). Memrnes were stripped nd re-proed for -ctin. (D) Normlized E-cdherin expression reltive to -treted cells tht were set to 1%. Vlues re the mensts.e.m. of three to six seprte experiments;, significntly different from control (P<.5). Lung denocrcinom cell lines expressed fmol ER/mg WCE protein, s mesured y specific [ 3 H]E 2 inding, nd no sttisticl difference ws detected etween cell lines from mles or femles (Fig. 3G). [ 3 H]E 2 inding in the lung smples ws significntly lower thn in cells. Since possile explntion for the lck of E 2 response in lung denocrcinom cells from mles is tht ER is excluded from the cell nucleus, we prepred NEs nd CEs nd mesured [ 3 H]E 2 inding to ech frction (Fig. 3H). [ 3 H]E 2 inding ws higher in NEs thn in CEs, ut no difference ws detected etween smples from femles or mles. Trnscriptionl ctivity of endogenous ER in lung denocrcinom cells A second function of ER, in ddition to E 2 inding, is to ctivte gene trnscription. Most responses to E 2 re medited y the genomic ER pthwy in which E 2 stimultes or inhiits the trnscription of trget genes (Klinge 2). To compre the trnscriptionl ctivity of endogenous ER in the denocrcinom cell lines, we performed trnsient trnsfection ssys using luciferse reporter driven y two tndem copies of n ERE (Klinge et l. 24). E 2 did not increse EREdriven luciferse ctivity in NF164 cells (Fig. 4A) or in,, or H1792 cells from mles (Fig. 4B E). Thus, similr to results seen in the prolifertion studies, the denocrcinom cell lines from mles were non-responsive to E 2 in these trnsient trnsfection ssys. ICI 182,78 suppressed sl trnscription in, ut not in ny other cell line from mles. Since ICI 182,78 trgets ER to the 26S protesome for degrdtion (Mrsud et l. 23), these results suggest possile role for ER in sl trnscription of the trnsfected ERE-luciferse reporter in cells. In contrst, E 2 stimulted ERE-driven luciferse ctivity in the denocrcinom cell lines from femles (Fig. 5) nd this stimultion ws inhiited y concomitnt tretment with or ICI 182,78, indicting tht the effect ws ER medited. Further, these results re concordnt with results from the MTT ssys indicting E 2 responsiveness in lung denocrcinom cell lines from femles ut not mles, despite equivlent ER nd ER protein expression nd specific [ 3 H]E 2 inding ctivity. nd ICI Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

14 S M Dougherty et l.: ER nd lung denocrcinom 182,78 suppressed sl trnscription in H1793 cells indicting tht, s reported y others, -occupied ER cn ctively suppress trnscription (Brslou et l. 1998, Zhng et l. 1998, Plns-Silv et l. 21, Thoms et l. 23). PR my e mrker of estrogen responsiveness in some lung denocrcinom cells from femles. PR is well-estlished mrker of estrogen responsiveness in rest cncer (McGuire et l. 1986). Reports on PR expression in NSCLC re mixed. Some prffin-emedded tumors showed PR expression y immunohistochemicl stining (Kiser et l. 1996, Su et l. 1996), ut other studies reported no PR expression (Di Nunno et l. 2, Rdzikowsk et l. 22). Although some studies found no correltion etween ER nd PR immunorectivity in NSCLC (Kiser et l. 1996, Su et l. 1996), one study reported higher PR in lung denocrcinom tumors from femle ptients compred with SCLC nd denocrcinoms from mle ptients (Kiser et l. 1996). To determine if E 2 stimulted PR expression in lung denocrcinom cell lines, cells were grown in DCC-FBS for 24 h nd then treted for 24 h with. All the lung cell lines expressed PR-B (Fig. 6A). The positive control cells expressed predomintely PR-B nd E 2 incresed PR protein expression y % (Fig. 6B). Interestingly, E 2 incresed PR expression in H1395 nd H1435 cells from femles. Thus, two out of five of the cell lines from femles showed E 2 -stimulted PR expression. E 2 decresed PR expression in nd cells from mles. We conclude tht PR my e n endogenous mrker for E 2 responsiveness in some lung denocrcinoms from femles. E-cdherin expression is not stimulted y E 2 in lung denocrcinom cell lines E-cdherin is clcium-dependent cell cell dhesion molecule tht is mrker for epithelil differentition nd is considered to e tumor suppressor gene (Merot et l. 24). The mrna nd protein levels of E-cdherin were recently reported to e stimulted y E 2 in 21T denocrcinom nd in 273T squmous cell crcinom lung cncer cell lines (Hersherger et l. 25). Thus, we investigted whether E 2 stimultes E-cdherin expression in the cell lines used in this study (Fig. 6C). As nticipted, E-cdherin ws not expressed in NF164 cells. E 2 decresed E-cdherin expression in cells (Fig. 6D). This grees with previous reports on E 2 ER-medited inhiition of E-cdherin expression in nd other rest cncer cell lines (Oesterreich et l. 23). E-cdherin ws expressed in NHBE cells nd its expression ws not ltered y E 2. Not ll the denocrcinom cell lines expressed E-cdherin nd in the cell lines expressing E-cdherin i.e.,, H1395, H1435, nd H273 E 2 either hd no effect or decresed E-cdherin expression (Fig. 6D). These results contrdict the conclusion tht E-cdherin is n E 2 -up-regulted trget gene in NSCLC cells (Hersherger et l. 25); however, ecuse different cell lines were used, no generliztions cn e mde. We suggest tht the cell lines expressing little or no E-cdherin represent more ggressive lung denocrcinom cell lines i.e.,, H1792 nd H1793 since functionl loss of E-cdherin is ssocited with n epithelil-to-mesenchyml trnsition in crcinogenesis tht results in invsion (Kumr & Hung 25). Indeed, is from lymph node metstses, nd H1792 from pleurl effusion from ptient with stge 4 disese; H273 is from ptient with stge 3A disese, ccording to the ATCC we site ( PBP/TRAP22/DRIP25 expression is higher in lung denocrcinom cell lines thn NHBE cells A difference in ER expression does not pper to e responsile for the prolifertive ctivity of E 2 nd the nti-prolifertive ctivity of nd ICI 182,78 in lung denocrcinom cells from femles versus mles. Thus, we hypothesized tht the expression or ctivity of some criticl component(s) of the ER signling pthwys, e.g. coctivtors nd/or corepressors, my e different etween lung denocrcinoms from mle nd femle ptients, possiility tht we re currently investigting. Previous studies showed tht ER intercts directly with coctivtor peroxisome prolifertor-ctivted receptor (PPAR)- inding protein (PBP)/thyroid receptor-ssocited protein (TRAP)22/Vitmin D receptor intercting protein (DRIP)25/Meditor 1 (MED1) nd tht DRIP25 is recruited to estrogen trget genes in n E 2 -dependent, cyclic mnner in rest cncer cells (Burkov et l. 2, 22, Kng et l. 22). Further, since PBP/TRAP22/DRIP25 is n essentil meditor of ER-medited trnscription t estrogen trget genes (Zhng et l. 25), we exmined the expression of DRIP25 protein in lung denocrcinom cells from femles versus mles (Fig. 7). DRIP25 expression ws higher in the lung denocrcinom cell lines nd in cells compred with NHBE or NF164 cells. The nture of the identity of the lower moleculr weight nds recognized y the DRIP25 ntiody in Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

15 Endocrine-Relted Cncer (26) DRIP25 α-tuulin DRIP25/α-tuulin H1793 is unknown, ut only the full-length DRIP25 vlue ws included in the clcultions (Fig. 7B). Although, H1435 cells (femle) showed expression similr to tht of (mle) nd (mle), the verge DRIP25 expression in lung denocrcinom cell lines from femles ws significntly higher thn tht in the cell lines from mles. Discussion NHBE NF164 H1435 DRIP25/ α-tuulin mle femle H1793 mle femle B NF164 NHBE H1435 H1792 H1793 H273 The mechnisms ccounting for the oserved higher incidence of lung denocrcinom in femles versus mles, whether smokers or non-smokers, re unknown, lthough there re epidemiologicl s well s iochemicl studies indicting tht estrogen my ply role in this difference (Stile & Siegfried 23). Here, we report tht lung denocrcinom cell lines from femle, ut not mle ptients proliferte in response to E 2 nd this prolifertion is locked y nd ICI 182,78, indicting tht the response is ER medited. These results re similr to E 2, nd ICI 182,78 responses in rest cncer A H273 NF164 H1792 kd Figure 7 Coctivtor DRIP25 is expressed in lung denocrcinom cell lines. Western lots for PBP/TRAP22/DRIP25 were performed using monoclonl ntiody to DRIP25 (Pined Torr et l. 24). The lots were stripped nd re-proed for -tuulin for normliztion. Vlues of pixel densities were normlized y cells, which were set to 1, nd re the verge of two lotsts.d. The inset shows the ments.e.m. for vlues from the denocrcinom cell lines from mles nd femles;, significntly different from the verge vlue from cell lines from mles (P<.5). cells (Bowers et l. 2). In ddition to E 2 -induced cell prolifertion, the cell lines from femles, ut not mles, showed trnscriptionl ctivtion of n ERE-luciferse reporter y endogenous ER in response to E 2. Surprisingly, despite the prolifertive nd trnscriptionl response to E 2 in femle versus mle denocrcinom cells, ER expression (s mesured y three independent tests) ws similr etween the genders. Rel-time PCR reveled tht mrna for ER in ll the lung denocrcinom cell lines ws expressed t level comprle with tht of ER in cells nd t higher level thn ER in or ny of the norml or neoplstic lung cells. There ws no gender-dependent difference in ER or ER mrna expression levels etween denocrcinom cells. Specific [ 3 H]E 2 inding nd the estimted expression levels of full-length ER nd ER, sed on immunolotting with known concentrtions of rher or rher s clirtors, were lso similr etween the genders. Thus, difference in ER expression does not pper to e the mechnism ccounting for the prolifertive ctivity of E 2 nd the nti-prolifertive ctivity of nd ICI 182,78 in lung denocrcinom cells from femles versus mles. Accordingly, we speculte tht the expression or ctivity of some criticl component(s) of the ER signling pthwys, e.g. coctivtors nd or corepressors, my e different etween lung denocrcinoms from mle nd femle ptients. Indeed, we oserved tht the verge expression of the coctivtor PBP/TRAP22/DRIP25, which is n essentil ER coctivtor tht intercts directly with ER nd components of the RNA polymerse II holoenzyme/meditor complex (Burkov et l. 2, 22, Shng et l. 2, Kng et l. 22, Sville et l. 22, Lee et l. 25, Zhng et l. 25), ws higher in lung denocrcinom cell lines from femles thn mles nd higher in the lung cncer cell lines compred with NHBE, NF164 or cells. PBP/TRAP22/ DRIP25 ws reported to e over-expressed in rest tumors nd cell lines reltive to norml humn mmmry glnd (Zhu et l. 1999) ut, to our knowledge, no one hs evluted its expression in lung denocrcinom. There is one report on coctivtor GRIP1 (TIF2) expression in NSCLC cell lines showing tht GRIP1 is expressed in denocrcinom nd T squmous cell crcinom cell lines, ut not in the 21T denocrcinom cell line (Hersherger et l. 25). Since there re currently 49 known ER-intercting coctivtors nd 18 known ER-intercting corepressors (Klinge 2, Smith & O Mlley 24), more experiments will e needed to ddress the possile different expression of ER coregultors in Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess

16 S M Dougherty et l.: ER nd lung denocrcinom lung denocrcinoms from mle nd femle ptients. Additionlly, non-genomic pthwys ctivted y ER lignds, such s the mitogen-ctivted protein kinse (MAPK)/epiderml growth fctor receptor (EGFR) pthwy, recently descried s eing E 2 ctivted in the 21T (Stile et l. 25) nd (Pietrs et l. 25) lung denocrcinom cell lines from mles, my lso ply role in the estrogen responses detected in our study. Clerly, this possiility will e nother importnt venue of further investigtion. We compred the ER expression in lung denocrcinom to tht mesured in other studies of lung cncer nd ER expression in rest cncer. To our knowledge, there is only one report tht estimted ER concentrtion in the denocrcinom cell line y [ 3 H]E 2 inding to e 1.9 fmol/mg cell memrne protein (Pietrs et l. 25) nd no reports of ER concentrtion in extrcts from humn lung tumors or lung cncer cell lines. In the lung denocrcinom cells, ER expression from Western lot estimtion using culovirus-expressed ER s clirtor were n verge of 1.54 nd 1.61 fmol/mg for mles nd femles respectively. The vlues for ER were the 3.98 nd 4.2 fmol/mg for mles nd femles respectively. In contrst, [ 3 H]E 2 inding ssys gve totl ER concentrtion s.14 nd.11 fmol/mg WCE for mles nd femles respectively. Western lot nlysis gve n estimte of 2 fmol ER/mg WCE using culovirus ER s stndrd. However, our HAP ssy dt indicte n verge [ 3 H]E 2 inding vlue of.15 fmol/mg WCE, which closely grees with the pulished vlue of.16 fmol ER/mg NE (Chloupk et l. 1992). This vlue grees with those for the NE from lung denocrcinom cells (Fig. 3). ER expression, s mesured y [ 3 H]E 2 inding, in rest tumors rnges etween 1 1 fmol/mg nucler protein (.1 1fmol/mg) nd 2 3 pmol/mg (2 3 fmol/mg) cytosolic protein (Forster et l. 24). The ltter vlues re greter thn the lung denocrcinom cells nd gree with the higher [ 3 H]E 2 inding tht we detected in the cells. We suggest tht the difference etween the western nd [ 3 H]E 2 inding estimtes of ER concentrtion indicte tht not ll of the expressed ER protein is le to ind lignd. From these vlues, it ppers tht only 1% of ER inds [ 3 H]E 2. Whether this is the result of cell extrction nd the ssy techniques or iologicl property of ER remins to e determined. Our results in lung denocrcinom differ from rest cncer where progression to estrogen independence nd nti-estrogen resistnce is often ssocited with decresed ER expression (Yng et l. 21); the results lso differ from endometril nd ovrin cncer where the ER :ER rtio is decresed compred with the respective non-cncer tissues (Pujol et l. 1998, Fujimoto et l. 2, Li et l. 23). Moreover, E 2 inding ws higher in NEs compred with CEs, indicting tht sequestrtion of ER outside the nucleus does not pper to e the cuse of the lck of ER responses in the denocrcinom cells from mles. Furthermore, gender hd no pprent effect on ER or ER expression. ER expression ws higher thn ER in ll denocrcinom cell lines. These dt gree with previous reports tht ER expression ws higher thn ER in (Stile et l. 22, Hersherger et l. 25) nd H1435 (Mollerup et l. 22) cell lines. To our knowledge, the only report of ER nd ER protein expression in norml humn lung, y immunostining, indicted overlpping s well s celltype-distinct ronchiolr expression of ech sutype (Tylor & Al-Azzwi 2). There re only three reports evluting the expression of ER nd ER proteins in lung denocrcinom tumors or cell lines, nd these reports reched different conclusions, especilly regrding ER expression (Omoto et l. 21, Fsco et l. 22, Hersherger et l. 25). There re mny more reports on ER nd ER mrna expression in norml humn lung nd NSCLC y RT-PCR (Fsco et l. 22, Mollerup et l. 22, Rdzikowsk et l. 22, Stile et l. 22). Notly, ER expression is highly vrile etween smples nd lortories, differences tht re proly due to the technique employed nd the method of smple preprtion. Although denocrcinom cells from femles nd mles hd comprle expression of ER, the ER ntgonist R,R-THC inhiited E 2 -induced cell prolifertion selectively in the femle cell lines. These cell lines did not hve lower ER :ER rtio versus cells from mles. Thus, the mechnism ccounting for this oservtion is unknown. Clerly further experiments to scertin the roles of ER nd ER in denocrcinoms from femle nd mle ptients is wrrnted. The inhiition of E 2 -induced prolifertion y R,R- THC ws similr to tht detected with ICI 182,78, ut less thn tht detected for, suggesting the possiility tht ER s well s ER my e involved in E 2 -induced cell repliction in these cell lines. lone inhiited denocrcinom cell prolifertion in cells from femles, ut the pure steroidl nti-estrogen ICI 182,78 lone inhiited only H1395 (femle) prolifertion. Both nd ICI 182,78 inhiited E 2 -induced cell repliction. In the two Downloded from Bioscientific.com t 9/19/218 6:53:53PM vi free ccess