The soy isoflavone genistein promotes apoptosis in mammary epithelial cells by inducing the tumor suppressor PTEN

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1 Crinogenesis vol. no.1 pp , 5 doi:1.193/rin/gi131 dvne ess pulition My 19, 5 The soy isoflvone genistein promotes poptosis in mmmry epithelil ells y induing the tumor suppressor huvnesh Dve 1,, Rene R.Eson 1, S.Renée Till 1, Yn Geng 1,, Mihel C.Velrde, Thoms M.dger 1, nd Rosli C.M.Simmen 1,, metolite(s) nd/or other systemi ftors indued y. 1 rknss Children s Nutrition Center nd Deprtment of Physiology nd iophysis, University of rknss for Medil Sienes, Little Rok, R 7, US To whom orrespondene should e ddressed t: rknss Children s Nutrition Center, 11 Mrshll Street, Little Rok, R 7, US. Tel: þ ; Fx: þ ; Emil: simmenrosli@ums.edu The isoflvone genistein (), iologilly tive omponent of soy foods, is ssoited with redued rest ner risk in women who onsume soy-rih diets. hs een reported to influene mny iologil proesses, of whih suppression of ell prolifertion nd stimultion of poptosis re onsidered to e the mjor pthwys underlying its inhiition of tumorigenesis. This study evluted the mehnism y whih diets ontining promote mmmry epithelil ell deth. We report tht mmmry glnds of young dult femle rts exposed from gesttion dy to postntl dy 5, to IN-93G diets ontining s sole protein soure, sein () supplemented with, or soy protein isolte (SPI þ ) hd inresed poptosis, reltive to rts fed diet devoid of. Mmmry glnd prolifertion ws unffeted y diet. The inresed poptoti index in mmmry glnds of nd SPI þ rts ws ompnied y inresed levels of the tumor suppressor protein (phosphtse nd tensin homolog deleted in hromosome ten), leit enhned mmmry expression of the pro-poptoti p1, x nd ok genes ws oserved only in -fed rts. -indued poptosis in MCF-7 ells ws onomitnt with inresed expression, nd this ws rogted y sirn. MCF-7 ells treted with serum from - or SPI þ -fed rts hd inresed poptosis s well s inresed levels of the trnsript. sirn ttenuted the inresed poptoti response of MCF-7 ells to serum from rts fed SPI þ or, lthough the inhiition to sl ( serum) poptoti levels ws hieved only for ells treted with serum. Deresed p1 nd ok gene expression ompnied the inhiition of poptosis y sirn. Dt implite in the indution of poptosis y nd suggest tht the promotion of poptosis leding to inhiition of tumorigenesis in vivo y diets ontining my lso involve the distint tivities of yet unknown revitions: IN, merin Institute of Nutrition;, sein; DM, 7,1 dimethylenz[]nthrene; GD, gesttion dy;, genistein; PCN, proliferting ell nuler ntigen; PI3-K, phosphtidylinositol-3-kinse; PND, postntl dy;, phosphtse nd tensin homolog deleted in hromosome ten; qpcr, quntittive rel-time polymerse hin retion; SEM, stndrd error of the men; sirn, smll interfering RN; SPI þ, soy protein isolte; TUNEL, terminl deoxynuleotidyl trnsferse-medited deoxy-utp nikend leling. Introdution rest ner ffets one of nine women in their lifetime nd is the seond leding use of ner-relted deths, next to lung ner, in the US femle popultion (1). Sine this is heterogenous disese tht hs een suggested to rise s result of oth geneti nd epigeneti modifitions ( ), there is no known single effetive strtegy for its prevention or tretment. However, diet hs een proposed to onstitute n importnt determinnt for deresing risk of rest ner (5 7). Evidene to support this inlude reent epidemiologil studies inditing tht the inidene of the disese is signifintly lower in sin women whose intke of soy produts, espeilly during dolesene, is 5 times more thn y merin femles (8,9). Soy produts re rih soure of phytoestrogens of whih the primry soy isoflvone, genistein (), is onsidered to ply key role in rest ner protetion (1 1). exhiits estrogen gonist nd ntgonist tivity through estrogen reeptor (ER)- nd ER- medited pthwys (13,1), tht is dose- nd ell ontext-dependent (15). It is lso n inhiitor of protein tyrosine kinses, whose tions re ruil to the ontrol of ellulr growth nd poptosis (1). More reent studies using nimls nd humn ells suggest tht multiple regultory pthwys ould e influened y, inluding through medition of stress responses (17); poptosis (18,19); HER-/neu () nd epiderml growth ftor reeptor (1) signling; nd proteosoml tivity (). Norml ells differ from ner ells in their ility to undergo poptosis when ellulr DN dmge is initited (3). Consequently, the geneti nd iohemil mehnisms tht onfer tivtion of the deth mhinery nd ssoited deth genes hve een the sujet of intense investigtions, in n effort to develop effetive strtegies for humn ner prevention nd therpy (,5). Studies from the lst few yers hve estlished ritil role for the dul lipid/protein phosphtse (phosphtse nd tensin homolog deleted in hromosome ten) in tumor suppression ( 9). Loss of or redution in expression hs een reported in wide rry of humn ners, inluding those of the uterus, prostte, lung, mmmry, nd ovry (3 3), nd is ssoited with poor linil outome (33). ordingly, in mouse models of defiieny, inresed suseptiility to mmmry neoplsi (3) nd preoious mmmry development (35) were oserved. The est eluidted funtion of s tumor suppressor reltes to its negtive regultion of the PI3- kinse/kt pthwy y virtue of its ility to dephosphorylte phosphtidylinositol 3,,5-triphosphte (3). Constitutive kt tivity s result of loss of funtion, leds to inresed prolifertion nd redued poptosis, oth of whih re hllmrks of tumorigeni potentil. Other reently defined Crinogenesis vol. no.1 # Oxford University Press 5; ll rights reserved Downloded from y guest on My 9, 13

2 .Dve et l. tumor-suppressive mehnisms of inlude inhiition of the growth ftor-tivted rs/mitogen tivted protein kinse pthwys (37), dephosphoryltion of fol dhesion kinse leding to inhiition of ell migrtion (38), nd reruitment of p53 in response to DN dmge (39). Our group nd others hve used rt models of hemillyindued mmmry tumorigenesis to evlute the effets of diet during erly development on dult risks of mmmry ner ( ). These studies demonstrted inhiition of DM- () nd NMU-indued (3) mmmry tumor development in rts lifetime-fed soy protein isolte (SPI þ ), mimi of the sin diet rih in isoflvones, reltive to those fed the ontrol sein () diet. The underlying mehnism(s) for these effets of SPI þ remins poorly defined, despite oservtions tht ltertions in differentition, prolifertion, poptosis, nd rinogen metoli pthwys my e involved (15, 7). Moreover, soy produts ontin host of yet unidentified peptides s well s non-protein omponents, inluding sponins, phyti id, lignns nd other phytohemils, whose individul nd umultive iologil effets re unknown. In the present study, we investigted the mehnism wherey the soy isoflvone exerts its protetive effets ginst mmmry tumorigenesis in vivo (1 1). We demonstrte tht enhnes mmmry ell poptosis y inresing the expression of the tumor suppressor/pro-poptoti protein s well s the pro-poptoti genes ok, x nd p1. We lso show tht soy, the nturl dietry soure of, mimis -indued poptosis, leit other signling pthwys in ddition to tht of, my e involved in this response. Further, we provide evidene for the regultion of systemi pro-poptoti ftors whose tions involve signling, y diets ontining. Our oservtions provide mehnisti insights into the role of dietry in the inhiition of mmmry tumorigenesis. Mterils nd methods nimls Proedures for niml re nd tretments followed the guidelines pproved y the University of rknss for Medil Sienes niml Cre nd Use Committee. Time-mted Sprgue Dwley rts, purhsed from Chrles Rivers Lortories, In. (Wilmington, M) were housed individully in polyronte ges under onditions of C, % humidity, nd 1-h light drk yle. Rts t gesttion dy (G-D) were rndomly ssigned to one of three semipurified isolori diets mde ording to the IN-93G formultion (8), with orn oil sustituting for soyen oil nd ontining s sole protein soure: (i) sein (; New Zelnd Milk Produts, Snt Ros, C); (ii) to whih ws dded genistein in the glyone form (, 5 mg/kg feed) (Sigm Chemil Co., St Louis, MO); nd (iii) soy protein isolte (SPI þ ; Sole Compny, St Louis, MO). The ltter ontins 39 1 mg totl isoflvones/kg diet, inluding 1 mg/kg genistein nd 1 mg/kg didzein, oth expressed s glyone equivlents (). nimls were provided food nd wter d liitum. t delivery, pups from dms of the sme diet groups were pooled nd ten pups (5 per sex) were rndomly ssigned to eh dm for sukling. Femle pups were wened t postntl dy (PND) 1 to the sme diets s their dms nd were fed the sme diets throughout the study. Mle pups were used in unrelted studies. Femle pups in ll three groups onsumed the sme mounts of diet nd did not differ in the durtion of their estrous yles (dt not shown). t PND5, femle pups were killed nd the dominl mmmry glnd (glnd numer ) pir ws removed. Portions of the left glnd were fixed for prffin emedding, while the right glnd ws immeditely homogenized in TriZol for RN extrtion. Immunohistohemistry Mmmry glnds were fixed overnight in 1% uffered-formlin, emedded in prffin, nd setioned. The proedures for ntigen retrievl in Citr Plus (iogenex, Sn Rmon, C), nd inution with loking solution (Cs 179 lok; Zymed, Sn Frniso, C) to minimize non-speifi inding were previously desried (9). Primry ntiodies used were nti-proliferting ell nuler ntigen (PCN, lone PC1; Dko Corp., Crpinteri, C), nd nti- (1; Snt Cruz iotehnology, In., Snt Cruz, C). Immunoperoxidse stining ws developed with 3,3 -diminoenzidine hromgen (Dko Corp.), nd slides were ounterstined with hemtoxylin. Four rndomly hosen fields () per slide per rt, with two slides for eh tissue lok from 5 individul rts per diet, were nlyzed for presene of drk rown olor-stining ells, inditing positive expression. The prolifertion index ws lulted s perent of PCN-positive ells reltive to the totl numer of ells ounted for eh mmmry struture (terminl end ud, TE; loules, LO; dutl epithelium, DE). (ytoplsmi nd nuler) expression ws determined y ounting the numer of positive ells per struture. Terminl deoxynuleotidyl trnsferse-medited deoxy-utp nik-end leling (TUNEL) ssy to detet poptoti ells ws performed following the mnufturer s instrutions (Onogene, L Joll, C), exept tht inution with TdT ws rried out for 1 h t 37 C. TUNEL-positive ells were ounted from three rndomly seleted fields t per slide, nd two slides were evluted for eh tissue lok from four individul rts per diet. RN extrtion nd quntittive rel-time RT PCR (qpcr) Totl RN ws isolted from mmmry glnds using TriZol regent (Invitrogen, Crlsd, C). Integrity of isolted RNs ws onfirmed using the RN Nno LChip kit with the gilent ionlyzer System (gilent iotehnologies, Plo lto, C). Totl RN (1 mg) ws reverse-trnsried using rndom hexmers nd multi-srie reverse trnsriptse in two-step RT PCR retion, following the mnufturer s instrutions (io-rd Lortories, Herules, C). Primers for PCR were designed to spn intron/ exon juntions (PrimerExpress; pplied iosystems, Foster City, C) to minimize mplifition of residul genomi DN. The primer sequenes for rt x, l, nd ok re (sense nd nti-sense, respetively): x (5 - GGCGTTGGCGTGC-3 ; 5 -GCTGCCCCGGGGC-3 ); l (5 -GGGGTTGTGGCCTTCTTTG-3 ; 5 -GCCGGTTCGGTC- TCGTCT-3 ), nd ok (5 -CGTCCCGCGTTTCGGT-3 ; 5 -CCCTGTGTCCTGCTGGG-3 ); the primer pir for p1 ws previously desried (9). PCR mix (5 ml) ontined optiml onentrtions (1 nm) of primers, 1 ng of DN, nd SYR Green PCR Mster Mix (io-rd Lortories). The onditions for qpcr nd determintion of reltive trnsript levels were desried previously (9). Reltive gene expression ws lulted with 18S rrn s the internl ontrol nd ws expressed s ritrry units. Cell ulture, tretments nd ssys Humn rest ner ells (MCF-7) were otined from merin Type Culture Colletion (TCC; Mnsss, V) nd ultured in DMEM ontining 1% (v/v) fetl ovine serum (FS) nd 1% ntiioti/ntimyoti solution (GICO, Crlsd, C) in 5% CO :95% ir t 37 C. For TUNEL, ells were seeded t density of 1. 1 ells per well on immunofluoresene hmer slides (Fisher Sientifi, In.). fter 1 h inution, ells were wshed with PS nd inuted in fresh DMEM ontining.5% FS. Tretments were ser pooled from PND5 rts lifetime-fed (n ¼ ), SPI þ (n ¼ ) or (n ¼ 7) dded t 1% finl onentrtion. Twenty-four hours lter, ells were rinsed with PS, fixed in % prformldehyde for 3 min, wshed with PS, nd suessively inuted with.1% Triton X-1/PS for 15 min nd Proteinse K for 5 min. Cells were inuted with fluoresein-leled TdT regent (Onogene) in humidified hmer t 37 C for 1 h. Leled nulei were ounted in three seprte fields (1) ontining 3 ells eh, using n Olympus I-71 mirosope with stndrd fluoresene filter. Dt re presented s the perent of leled nulei from the totl numer of ells ounted. The isoltion nd ulture of mmmry glnd epithelil ells followed protools desried y Jeffrey Rosen s lortory ( protools) (ylor College of Mediine, Houston, TX), s dpted from n initil report y Pulln nd Streuli (5). The dominl mmmry glnd pir (numer ) from PND8 rts exposed to diet eginning t gesttion dy ws used s soure of epithelil ells. Mmmry tissues from two rts were pooled, nd equl liquots of isolted ells were plted in six-well ulture dishes in F1 medium ontining gentmiin (1 mg/ml), insulin (1 mg/ml), hydroortisone ( mg/ml), epiderml growth ftor (1 ng/ml) nd units of peniillin/streptomyin. fter 8 h, the medium ws hnged to F1 ontining 5% FS nd the ove ftors t one-hlf of their originl onentrtions. Cells were llowed to grow for nother 3 dys efore tretments with, SPI þ or ser (1% finl onentrtion) s desried for MCF-7 ells, nd were nlyzed for gene expression. Experiments were repeted three times. Downloded from y guest on My 9, 13

3 in genistein-indued poptosis Trnsfetion of smll interfering RNs (sirns) MCF-7 ells were seeded t density of ells per well in -well ulture pltes nd llowed to grow to 5 7% onfluene prior to trnsfetion. Cells were trnsfeted with duplexed sirn (1 nm) ginst humn (sense: 5 -GCUUGGGCGUUCGtt-3 ; nti-sense: 5 -CUGUU- CGCCUUCGUCtt-3 ; mion, In., ustin, TX) using Lipofetmine (Invitrogen) in DMEM ontining 1% FS. To evlute the speifiity of the sirn trnsfetion proedure, MCF-7 ells were trnsfeted in prllel with duplexed sirn ginst GPDH (mion) or n irrelevnt sirn (mion), s positive nd negtive ontrols, respetively. fter inution for 5 h t 37 C, ells were treted with pooled ser from PND5 rts fed, SPI þ or (see ove), dded to finl onentrtion of 1%. Control ells reeived 1% FS in lieu of rt serum. Cells were inuted for n dditionl h nd then proessed for TUNEL ssy s desried ove. nother set of ells ws similrly treted with pooled, SPI þ or ser (1% finl onentrtion), fter trnsfetion with sirn (1 nm) or fter mok-trnsfetion. Cells were olleted in TriZol regent h lter. RN extrtion nd nlysis of trnsript levels y qpcr followed the protools desried ove. Humn primers for PCR re (sense nd nti-sense, respetively): (i) x (5 -CTGGGCTGCGGGTGT-3 ;5 -TCGC- TGCCCTCGG-3 ); (ii) p1 (5 -CGCGCCTTCCTCTCCC-3 ; 5 -GGCGGGCCGGC-3 ); (iii) (5 -GCTTGGGTTT- CCTGCG-3 ;5 -GGCGGTGTCTTGTCTTTC-3 ); nd (iv) ok (5 -TCTTCTCTGCGGCTCCG-3 ;5 -CCCCGCTCGGGCC-3 ). 18S rrn ws used s the normliztion ontrol, nd levels of RN trnsript were expressed s ritrry units. Sttistil nlysis Dt from in vitro studies were derived from t lest 3 independent experiments nd re presented s lest-squres mens SEM. Differenes etween diet groups were sttistilly nlyzed y one-wy or two-wy NOV, followed y inspetion of ll differenes etween pirs of mens y Tukey s test. P.5 ws onsidered sttistilly signifint. Results Diet effets on mmmry prolifertion nd poptosis The prolifertive index of mmmry strutures from PND5 rts lifetime exposed to, SPI þ nd diets ws determined y PCN stining of histologil setions. s shown in Figure 1, the ptterns of ell prolifertion were similr for mmmry glnds of nd SPI þ rts, with TE showing greter PCN immunoretivity thn LO (, P ¼.; SPI þ, P ¼.1) nd DE (, P 5.1; SPI þ, P ¼.). The prolifertion pttern for group differed slightly, with PCN immunoretivity eing highest in TE nd LO, nd lowest in DE (P 5.5). For eh mmmry struture type, prolifertion index ws not ffeted y diet. In ontrst, there ws signifint inrese (P 5.5) in the numers of poptoti (TUNEL-positive) ells in mmmry strutures of SPI þ nd, reltive to those of rts (Figure 1). However, while the numers of TUNEL-positive ells in TE were omprle for rts fed SPI þ nd diets, LO nd DE strutures exhiited higher numers of poptoti ells (P 5.5) in thn in SPI þ rts. Diet effets on mmmry expression Given the role of the dul protein/lipid phosphtse in poptosis (,3), tht loss of expression is ssoited with inresed tumorigenesis in tissues inluding the mmmry glnd (31,33), nd tht muttions in the gene re linked to 3% lifetime risk of rest ner (51), we evluted the levels of protein in mmmry strutures s funtion of diet. Mmmry tissue setions were inuted with nti- ntiody to quntify the numers of immunopositive ells. ws immunololized to ytoplsmi nd nuler omprtments in TE, LO nd DE strutures of mmmry glnds (Figure ). The numer of immunopositive ells in TE did not differ with % PCN-Positive Cells poptoti Cells / Struture TE LO DE Diet diet (Figure ). LO nd DE of rts fed SPI þ nd hd inresed numers of immunopositive ells thn in those fed (P 5.5), lthough for oth struture types, these numers did not differ etween SPI þ - nd -fed rts. Effet of diet on mmmry gene expression Sine SPI þ nd similrly indued mmmry ell poptosis in vivo (Figure 1) onomitnt with inresed expression in LO nd DE (Figure ), we next evluted if the underlying poptoti signling pthwy(s) downstrem of ws similr for oth diets. To ddress this, we determined the expression levels of the poptoti-ssoited genes x, l, p1 nd ok in mmmry tissues of PND5 rts lifetime exposed to, SPI þ or diets. The mmmry expression of ll four genes did not differ etween the nd Diet TE LO DE Fig. 1. Cell prolifertion nd poptoti sttus of mmmry glnds of PND5 rts lifetime exposed to dietry, SPI þ nd. () PCN immunostining is expressed s perent of the totl numer of TE, LO nd DE ells ounted for given field. Dt re mens SEM, with two setions evluted for eh of n ¼ rts per diet group nd were nlyzed y one-wy (within eh diet) nd two-wy (diet y struture intertion) NOV. () The numers of stined nulei in TE, LO nd DE strutures from three rndomly seleted fields per slide, with two setions evluted for eh of n ¼ rts per diet group re presented s mens SEM. Dt were nlyzed y one-wy NOV. For eh struture, mens without ommon letter differ (P 5.5) Downloded from y guest on My 9, 13

4 .Dve et l. TE LO DE No d e f positive ells/struture TE LO DE Fig.. expression in mmmry strutures of PND5 rts lifetime exposed to dietry, SPI þ nd. -positive ells in TE, LO nd DE were identified y immunohistohemistry using nti- ntiody. () Representtive mirogrphs of TE, LO nd DE of PND5 rt mmmry glnds without ( ) nd with (d f ) immunostining for. Smll rrows ¼ ytoplsmi stining; Lrge rrows ¼ nuler stining. () The totl numer of immunopositive ells in nuler nd ytoplsmi omprtments ws ounted from four rndomly seleted fields per slide, using two slides per tissue lok from n ¼ rts per diet groups. For eh struture, mens without ommon letter differ (P 5.5), s nlyzed y one-wy NOV. See online Supplementry mteril for olor version of this figure. Diet Downloded from y guest on My 9, 13 SPI þ groups (Figure 3). y ontrst, mmmry expression of p1 (P ¼.1), x (P ¼.), nd ok (P ¼.3) genes ws higher for thn for rts (Figure 3), while tht for l ws omprle (P ¼.1) etween the two diets. Indution of mmmry poptosis y genistein involves We next used the mmmry epithelil rinom MCF-7 ells s n in vitro system to evlute diret role for in mediting the oserved indution of mmmry epithelil ell poptosis y in vivo (Figure 1). In this study, RN interferene (5) ws used to inhiit the expression of, nd resultnt effets on -indued poptosis were evluted y TUNEL. Two experiments were performed to initilly define the optiml onditions for the interferene ssy. First, GPDH sirn ws used s positive ontrol for the trnsfetion protool. The roust knok-down in GPDH mrn 179 expression (8%), s shown in Figure, onfirmed the working onditions for sirn trnsfetion (see Mterils nd methods). Under these onditions, sirn, when dded t 1 nm finl onentrtion suppressed sl expression y 7% (Figure ); hene, this sirn onentrtion ws used in susequent experiments (elow). Seond, ontrol sirn (designted negtive ontrol) ws ssessed for its ility to inhiit oth GPDH nd expression; its lk of effet on the expression of oth genes indited its utility s negtive ontrol for the poptosis experiments (Figure nd ). MCF-7 ells were treted with pure in the presene nd sene of trnsfeted sirn, nd expression levels were determined in RNs prepred from these ells. ( mm) inresed trnsript levels, nd this effet ws lost when ells were inuted with sirn during tretment (Figure 5). The levels of poptosis in -treted

5 in genistein-indued poptosis Gene Expression (ritrry Units) 8 SPI + GPDH (ritrry Units) p1 x l OK * 1 Control sirn Negtive Control 7 1 Gene Expression (ritrry Units) p1 x l OK Fig. 3. Expression of poptosis-ssoited genes in mmmry glnds of PND5 rts lifetime exposed to dietry, SPI þ nd. Levels of RN (mens SEM, representing n ¼ nimls per diet group) were mesured y qpcr nd normlized to 18S RN. The nlysis ws rried out seprtely for versus SPI þ () nd versus (). P 5.5 reltive to group for eh gene, when ompred y t-test. ells were lso ompred in the presene nd sene of trnsfeted sirn. There ws dose-dependent inrese in the perent of poptoti ells when ws dded t inresing onentrtions to ultures (1 mm mm ; P 5.1) (Figure 5). ddition of sirn rogted the poptosis-induing effet of in these ells. Effets of rt ser on mmmry ell poptosis nd gene expression To investigte whether indution y of mmmry poptosis in vivo ours through other mehnism(s) in ddition to its diret effet on mmmry epithelil ells, MCF-7 ells were ultured in the presene of serum (1% finl onentrtion) pooled from PND5 rts (, n ¼ ; SPI þ, n ¼ ;, n ¼ 7) lifetime exposed to dietry, SPI þ or, nd the numers of poptoti ells were mesured y TUNEL. s shown in Figure, the perent of poptoti ells ws signifintly inresed y 5- to -fold (P 5.5) with SPI þ or serum, reltive to serum. expression ws lso higher in MCF-7 ells grown in the presene of SPI þ (P ¼.8) nd (P 5.5) serum thn in ells grown in serum (Figure ). similr trend in the indution of gene expression ws oserved when primry ultures of rt mmmry epithelil ells were treted with serum from, SPI þ nd -fed rts (Figure ). nlysis of totl onentrtions in serum smples indited levels of mm,..1 mm nd undetetle for rts on, SPI þ nd diets, respetively (53). * * (ritrry Units) Control -medited effets on serum-indued mmmry ell poptosis nd gene expression The ove dt indite tht serum ontining levels in the rnge of 15 nm (present in 1% serum) n indue poptosis to the sme extent s mm of pure (Figure 5). To exmine whether is involved in poptosis indued y ftors present in serum, MCF-7 ells were treted with serum from, SPI þ or -fed rts, eh dded to finl onentrtion of 1%, in the presene nd sene of sirn. Consistent with the erlier results (Figure, ove), (P 5.1) nd SPI þ (P 5.1) serum inresed the perent of poptoti ells, reltive to serum (Figure 7 nd ). serum lone hd higher pro-poptoti tivity thn 1% FS. Cells trnsfeted with sirn (1 nm) nd inuted with the test serum (, SPI þ nd ) hd signifintly redued numers of poptoti ells thn serumtreted, mok-trnsfeted (without sirn) ells (P ¼.5 for ; P 5.1 for oth SPI þ nd ) (Figure 7 nd ). Interestingly, while sirn ompletely olished the pro-poptoti effet of serum to the level of serum, this ws not oserved for SPI þ serum. In the ltter, the perent of poptoti ells in ultures with dded sirn ws signifintly higher (y 5%) thn in those * sirn Negtive Control Fig.. Effet of speifi sirns to GPDH () nd () on gene expression. MCF-7 ells were trnsfeted with the indited sirns (1 nm finl onentrtion), nd the level of expression of eh gene ws quntified y qpcr. Vlues were normlized to tht of 18S rrn. Negtive ontrol represents n irrelevnt sirn tht is not speifilly trgeted to ny mrn sequene, nd ontrol represents mok-trnsfeted ells. Results re from one (GPDH sirn) nd two ( sirn) independent experiments, with two replites rried out per experiment. P 5.5, ompred with ontrol ells, y t-test in () Downloded from y guest on My 9, 13

6 .Dve et l. (ritrry Units) Ser sirn (µm) Ser sirn (µm) % poptoti Cells inuted with serum lone. Trnsfetion with the irrelevnt (negtive ontrol) sirn hd no effet on poptosis for ny of the tretments (dt not shown). Next, we determined whether disruption of expression resulting in ttenution of serum ( nd SPI þ )-indued poptosis, ltered the expression levels of the pro-poptoti genes x, p1 nd ok in serum-treted MCF- 7 ells. Consistent with their effets on poptosis, serum from rts fed nd SPI þ inresed the expression of the propoptoti genes p1 (effet of SPI þ ; P 5.1) nd ok (effet of ¼ SPI þ ), reltive to serum, leit the expression levels of x did not similrly hnge with these tretments (Figure 8). Suppression of expression y sirn resulted in diminished p1 expression, with greter effets noted in ells treted with nd SPI þ serum, thn in those treted with serum. Expression of ok ws lso inhiited y loking funtion under ll tretments. y ontrst, sirn hd no effet on x gene expression in ells treted with nd serum, ut inresed levels of x trnsripts in ells treted with SPI þ serum (Figure 8). Disussion The ellulr nd moleulr mehnisms underlying the protetive effets of soy-sed diets on hormone-relted ners, FS FS FS FS FS FS FS FS Fig. 5. Effet of on MCF-7 expression nd poptosis in the presene nd sene of sirn. () MCF-7 ells were trnsfeted with sirn (1 nm) nd inuted in FS (1% finl onentrtion) with or without dded ( mm). trnsript levels were quntified y qpcr; P 5.5 for mens with different letters, when nlyzed y one-wy NOV. () MCF-7 ells were treted with ( or 1 mm) in the presene nd sene of sirn (1 nm). poptoti index is expressed s perent of TUNEL-positive ells over the totl numer of ells ounted (verge of 3 per given field), with three fields evluted per slide, nd three slides evluted per tretment. Dt re mens SEM; P 5.5 for mens with different letters, when nlyzed y two-wy NOV % poptoti ells mrn (ritrry Units) MCF-7 Serum (1%) MEC inluding rest ner remin unler. Dt from in vivo nd in vitro studies hve linked, the primry soy-derived isoflvone to this ner protetion vi its ility to indue trget ell poptosis (1,18), dysregultion of whih is hllmrk of tumor ells nd underlies tumor progression, metstsis nd ggressiveness. In the present study, we demonstrted the poptoti-indutive properties of dietry soy protein (SPI þ ) nd its ssoited isoflvone genistein (), the ltter when onsumed s dietry supplement, on mmmry glnd strutures in vivo nd defined novel signling pthwy y whih indues poptosis for mmmry tumor protetion. We found tht mmmry epithelil ells, when exposed to SPI þ nd in vivo or to ser derived from rts fed these diets in vitro hd inresed expression, oinident with their inresed poptoti sttus. Suppression of expression in vitro olished poptosis indued y ser in the mmmry epithelil ell line MCF-7, ut did not totlly inhiit tht indued y SPI þ ser. Results suggest tht while predominntly utilizes the signling pthwy for indution of ellulr poptosis in mmmry epithelil ells, dditionl pthwys, possily engged y ^ MCF-7 Fig.. Effet of serum from PND5 rts lifetime exposed to dietry, SPI þ nd on MCF-7 ell poptosis nd gene expression. () MCF-7 ells were inuted for h with pooled serum from rts of the three diet groups, nd poptoti ells were visulized using fluoresein-leled TdT regent. poptoti index is expressed s perent of TUNEL-positive ells over the totl numer of ells ounted (verge of 3 per given field). Results (mens SEM) re from three independent experiments, with two replites rried out per experiment. Mens without ommon letter differ (P 5.5). () Primry ultures of rt mmmry epithelil ells (MEC) or MCF-7 ells treted s indited ove were nlyzed for gene expression y qpcr. Mens SEM re from three independent experiments. P 5.5 for mens with different letters; supersript â refers to P ¼.8, reltive to. Dt were nlyzed y one-wy NOV. Downloded from y guest on My 9, 13

7 in genistein-indued poptosis right Field SERUM SPI + SERUM SERUM % poptoti Cells d Ser FS FS SPI + SPI + RN sirn Fig. 7. poptoti sttus of MCF-7 ells treted with serum (1% finl onentrtion) from PND5 rts lifetime exposed to dietry, SPI þ or, in the presene nd sene of trnsfeted sirn (1 nm). () Representtive fields showing poptoti ells in mok-trnsfeted ( sirn) nd sirn-trnsfeted (þsirn) MCF-7 ells, treted with rt serum from the three diet groups. () The numer of poptoti ells for eh diet group ws ounted from n verge of 3 ells per field, with 3 fields evluted per slide, nd 3 slides evluted per tretment group. Dt re mens SEM; P 5.5 for mens with different letters, when nlyzed y two-wy NOV. See online Supplementry mteril for olor version of this figure. Downloded from y guest on My 9, 13 other soy-ssoited omponents ontriute to the poptoti tivity of dietry soy. Here we hve estlished the relevne of in the propoptoti tivity of on mmmry epithelil ells. We showed tht the mmmry strutures LO nd DE from young dult (PND5) rts lifetime exposed to diet hd higher levels of the protein nd onomitntly, inresed poptoti sttus, reltive to those fed the ontrol diet. We further showed tht pure s well s ser from rts lifetime exposed to -ontining diets indued expression nd lso poptosis in MCF-7 ells, mimiking tht oserved in vivo. In ddition, we demonstrted tht indution of poptosis in MCF-7 ells y or ser ws rogted y sirn direted ginst. Finlly, interferene of expression in vitro resulted in inhiition of ser-indued expression of the pro-poptoti genes p1 nd 1799

8 .Dve et l. (ritrry Units) p1 (ritrry Units) x (ritrry Units) ok (ritrry Units) d e d Downloded from y guest on My 9, 13 Ser sirn SPI + SPI Fig. 8. Effet of sirn on poptoti gene expression y MCF-7 ells treted with serum (1% finl onentrtion) from PND5 rts lifetime exposed to dietry, SPI þ or. The expression levels of, p1, x nd ok were determined y qpcr. Dt re mens SEM from three independent experiments; P 5.5 for mens with different letters, when nlyzed y two-wy NOV. ok, whose mmmry expression in vivo re enhned y dietry. These results indite tht tivtion y my e essentil for poptosis indution in speifi mmmry glnd strutures (LO nd DE) in young dult rts. The lk of similr usl reltionship etween expression nd poptosis in TE, the strutures most sensitive to hemil rinogenesis t the ge exmined here, is unler t the present time nd will require further studies. However, this my e relted to the differentition sttus of the TEs, reltive to LOs nd DE t PND5; the phosphoryltion sttus of protein, whih hs een shown to inhiit its tivity (5) in TEs; nd the ft tht regultion of poptosis is omplex, ontext-dependent proess involving signling pthwys other thn. The mehnism(s) y whih indues expression leding to poptosis is presently unler. However, this is 18

9 in genistein-indued poptosis proly unrelted to s funtion s generl inhiitor of tyrosine-speifi protein kinses sine the ltter tivity of ws shown to e effetive only t high doses (1 mm) (1), while the oinident indution of expression nd poptosis in vivo nd in vitro reported here ourred t 5-fold lower onentrtions. Consistent with this, the inhiition of NMU-indued rt mmmry tumorigenesis y ws reported to e independent of its inhiition of tyrosine kinse tivity (55). Studies to delinete whether modultes trnsription of the gene re ongoing in our lortory. The reent report of s intivtion of the nti-poptoti trnsription ftor NF-k y inhiiting the phosphoryltion of kt (19) is onsistent with the oserved indution of expression reported here. dephosphoryltion of phosphtidylinositol 3,,5-triphosphtes leds to negtive regultion of the PI3-K pthwy, resulting in redued kt phosphoryltion (3). Interestingly, the inhiition of NF-k s well s kt kinse tivities in the erlier study ws noted t 5 mm, in ontrst to the muh lower effetive in vivo onentrtion for indution of poptosis nd expression reported here, sed on the serum levels of found in rts fed these diets (SPI þ,. mm;, 1.7 mm). Further studies to orrelte the temporl nd dose-dependent effets of on indution nd kt intivtion re needed to ddress this pprent disrepny. Interestingly, NF-k hs een demonstrted to inhiit gene expression (5); thus,, kt, nd NF-k proly onstitute tightly regulted loop for the mintenne of ell poptosis nd homeostsis, nd whih is influened y nutrition. We hve determined tht PND5 rts lifetime exposed to dietry nd SPI þ differed in their men serum genistein onentrtions, despite omprle mounts of glyone equivlents in these diets (1 versus 5 mg/kg SPI þ nd diet, respetively). This pprent disrepny is proly relted to the moleulr forms of dietry in SPI þ (gluoronide nd sulfte onjugtes; free nd protein-ound glyones) versus the supplement (free glyone) (57). There is lso the possiility tht sorption nd proessing of soy protein-ssoited is signifintly different from pure. Nevertheless, sine the serum levels of determined for rts on SPI þ diet re similr to those previously reported for humns regulrly onsuming soy produts (.3. mm) (58), the oserved indution of mmmry epithelil ell poptosis y soy demonstrted in femle rts in the present study my hve iologil relevne in humn femles. It is worth noting tht while serum from rts fed nd SPI þ indued omprle responses in gene expression nd poptosis in MCF-7 ells, mmmry glnds from -fed rts hd greter poptosis nd enhned expression of the propoptoti genes p1, x nd ok, reltive to those fed SPI þ. Severl possiilities my explin these oservtions. First, other soy-ssoited omponents my negte nd/or modify the poptoti tivity of present in soy. Seond, mmmry stroml ells in vivo my serve s trgets for nd/or modify the epithelil ell tions of, other soy-ssoited iotive omponents, resulting in ltered stroml/epithelil ell intertions tht re importnt for in vivo epithelil ell response. Lst, expression in the stroml omprtment, muttions of whih hve een reported in rest rinom (3) my lso medite the poptoti response of mmmry glnd to ; however, we did not oserve signifint differenes in stroml expression s funtion of diet (dt not shown). lthough further experiments re required to evlute these possiilities, our oservtions of the distint iologil effets of in vivo when tken s dietry supplement versus when onsumed s norml onstituent of soy foods, rise questions on how the signling pthwys indued y nutritionl ftors present in foods my ollorte for optiml helth enefits. The present findings provide the groundwork for ddressing two importnt questions relted to diet nd dult risk of mmmry ner. One reltes to the underlying mehnism for the pro-poptoti effets of dietry SPI þ nd in vivo, whih do not pper to e dependent solely on irulting levels. We hve found tht SPI þ nd serum exhiit omprle poptoti tivities, despite their distint levels of (3-fold higher in thn in SPI þ ). Moreover, the poptoti tivities of nd SPI þ ser in vitro (with levels in the rnge of 15 nm, when serum ws dded t 1% finl onentrtion) were higher thn tht of pure used in the mm rnge. Given tht t lest 15% of ll ners re now onsidered to e used y inflmmtion (59,); tht ytokines re powerful modultors of the immune system (1), nd tht NF-k, n inflmmtion-indued trnsription ftor entrl to the inflmmtory response, is downregulted y (), possily through intivtion of kt, n nlysis of the differentil expression of NF-k-regulted pro-inflmmtory nd immunosuppressive ytokines in serum from SPI þ nd rts, reltive to those of rts, my provide importnt insights into the mehnisms of tion distint from those of SPI þ. More importntly, suh findings my suggest dietry mnipultion of the immune system s vile lterntive for ner protetion (). The other question reltes to the nture/linege of the mmmry ell popultions in vivo tht re highly sensitive to poptosis indued y dietry intke of SPI þ nd. It hs een suggested tht popultion of undifferentited ells with high tumorigeni potentil is present in the developing mmmry glnd, nd tht removl of these ells t n erly developmentl period ould led to mmmry phenotypes tht re less suseptile to geneti muttions t lter life stge (3,). It is tempting to speulte tht the few numers of ells indued to undergo poptosis y dietry SPI þ nd represent progenitor ells tht re trgets for trnsformtion during mmmry tumorigenesis, nd tht their preise trgeting my e relted to inresed expression. The pplition of epithelil progenitor ell mrkers suh s kertin nd S-1 (5) to identify the ell popultions trgeted for poptosis y dietry SPI þ nd should enle exmintion of this possiility. In summry, we hve presented evidene of role for in the indution of mmmry epithelil ell poptosis y. We further show tht the signling pthwy for tivtion of poptosis y dietry SPI þ my e mnifesttion of the umultive effets of other soy omponents, in ddition to -medited tion. Our findings provide frmework for funtionl dissetion of the involvement of in mmmry tumor protetion s medited y dietry ftors, nd present new insights onerning the regultion of systemi ftors y diet tht ould led to new pprohes for ner tretment nd prevention. Supplementry mteril Supplementry mteril is ville online t: oxfordjournls.org/. 181 Downloded from y guest on My 9, 13

10 .Dve et l. knowledgements The uthors thnk the following for their ontriutions to this study: Dr Frnk.Simmen for helpful disussions nd ritique of the mnusript, Mtt Ferguson nd Tmmy Dllri for niml work nd Pmel Tredwy for dt olletion. Funds for this reserh were provided y the USD-CRIS S to the rknss Children s Nutrition Center. Conflit of Interest Sttement: None delred. Referenes 1. Jeml,., Murry,T., Smuels,., Ghfoor,., Wrd,E. nd Thun,M.J. (3) Cner sttistis. C Cner J. Clin., 53, 5.. Tisty,T.D., Crwford,Y.G., Holst,C.R., Fordye,C.., Zhng,J., MDermott,K., Kozkiewiz,K. nd Guthier,M.L. () Geneti nd epigeneti hnges in mmmry epithelil ells my mimi erly events in rinogenesis. J. Mmmry Glnd iol. Neoplsi, 9, Nthnson,K.L., Wooster,R. nd Weer,.L. (1) rest ner genetis: wht we know nd wht we need. Nt. Med., 7, Jones,P.. nd ylin,s.. () The fundmentl role of epigeneti events in ner. Nt. Rev. Genet., 3, rker,D.J.P. (1997) Mternl nutrition, fetl nutrition, nd disese in lter life. 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