TGF-b1 actions on FRTL-5 cells provide a model for the physiological regulation of thyroid growth

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1 Oncogene (1998) 16, 1455 ± Stockton Press All rights reserved 0950 ± 9232/98 $12.00 TGF-1 ctions on FRTL-5 cells provide model for the physiologicl regultion of thyroid growth Crmen Crneiro 1, Clr V Alvrez 1, Jun Zlvide 1, Anxo Vidl 1 nd Fernndo DomõÂ nguez 1,2 1 Deprtmento de FisiologõÂ, Lortorio de Neurocienci `RmoÂn DomõÂnguez', nd 2 Unidd de Medicin Moleculr, Sntigo de Compostel, Spin Little is known out the TGF-1 mechnism tht promotes thyroid cell growth rrest. We ssessed TGF1 e ects on Fisher rt thyroid cell line (FRTL-5). This llowed us to study TGF-1 ction on thyroid cells in vrious physiologicl situtions such s ctively proliferting cells, resting cells stimulted to proliferte y the ction of vrious mitogens, nd resting cells. TGF-1 rrested proliferting FRTL-5 cells, incresing c-myc mrna levels nd reducing p27-free protein levels, without ecting either the cellulr content of p27 or the -p27 complexes. Moreover, TGF-1 tretment reduced the ctivity of cyclin E-CDK2 complexes nd, consequently, prb ws found to e hypophosphorylted. TGF-1 prevented resting cells to enter in the cell cycle when stimulted with growing medium (neworn clf serum plus mixture of ve hormones) ut not when TSH (thyroid stimulting hormone) plus IGF-1 (Insulin-like growth fctor I) were used s mitogens. Both stimuli incresed the levels of cyclins D1, D3 nd E ut TGF-1 hd greter e ect in decresing these cyclin levels in growing-medium stimulted cells thn in TSHIGF-1. This suggests tht for FRTL-5 cells, the content of these cyclins must exceed threshold to progress through the cell cycle. TGF-1 induced poptosis in quiescent cells, ccompnied y reduction in p27 protein levels nd n increse in c-myc expression. Interestingly, TGF-1-induced vritions in prothymosin lph nd c-myc mrna levels were not correlted. TGF-1 lwys promoted n increse of p15 mrna levels. In summry, our results point to the fct tht TGF-1 could ply physiologicl role in the control of thyroid growth through the modi ction of cell cycle regultory proteins. Keywords: poptosis; cell cycle; cyclin; thyroid; TGF1; p27 Introduction Tight regultion of cell numer is essentil for the mintennce of tissue homeostsis. In mny physiopthologicl circumstnces, growth ctivting nd inhiiting stimuli must e perfectly lnced in order to chieve the right mount of cell prolifertion. One exmple is thyroid hyperplsi in goiter development during which thyroid folliculr cells proliferte in response to goitrogen. Once the thyroid hs reched Correspondence: CV Alvrez Received 20 My 1997; revised 17 Octoer 1997; ccepted 17 Octoer 1997 certin size, further increse is stopped despite continuous dministrtion of the goitrogen. This is ccompnied with TGF-1 eing widely distriuted within the entire folliculr epithelium, while in norml thyroid it is restricted to prfolliculr C cells (Logn et l., 1994). After goitrogen withdrwl there is regression of the thyroid glnd medited in prt y poptosis (Dumont et l., 1992). This process is ccompnied y high levels of TGF-1 expression, suggesting tht it plys role in thyroid poptosis. TGF-1 strongly inhiits prolifertion in epithelil, endothelil nd lymphohemtopoietic cells. Although moleculr mechnisms tht medite this inhiition seem to e di erent depending on the cell type studied, TGF-1 cts y modifying the levels of cell cycle regultory proteins such s cyclin D, cyclin E nd CDK4 or through the ction of cyclin-cdks complexes inhiitors like p27, p15 or p21 (Sltis, 1996). An ction of TGF-1 on the tumour suppressor genes prb (Liho et l., 1990; Geng nd Weinerg, 1993) nd p53 (Ewen et l., 1995; Blydes et l., 1995) hs lso een descried. Moreover, such mechnisms re dependent on the prolifertive stte of ech speci c cell. For exmple, when serum-strved HCT kertinocytes re stimulted with serum, TGF-1 decreses CDK4 mrna levels (Geng nd Weinerg, 1993). Nevertheless, TGF-1 does not ect CDK4 mrna levels of exponentilly growing HCT cells (Hnnon nd Bech, 1994). Similrly, TGF-1 decreses c-myc levels in serum-strved ut not in proliferting mouse rolsts (Jnsen-Jurr et l., 1993). In contrst to the nding in epithelil cells, TGF-1 cn induce prolifertion in certin mesenchyml cells (Alexndrow et l., 1995; Mchwte et l., 1995). In the C3H 10T1/2 mouse rolsts, reduction on p27 protein levels seems to ply n importnt role in this induction (Rvitz et l., 1995). In some cell types, TGF-1 induced growth rrest is ccompnied y progrmmed cell deth (for review see Lieermn et l., 1995) while in other cses memers of the TGF- superfmily cn protect ginst poptosis (Krieglstein et l., 1995). The pthwys through which TGF- exerts these e ects re uncler due to the sence of model in which the growth inhiitory ction could e dissected from the poptotic e ect. FRTL-5 is rt epithelil folliculr cell line tht retins mny of the chrcteristics of norml thyroid cells. They hve TSH-dependent growth, synthesize thyrogloulin, form follicle-like structures nd re nontumorigenic (Amesi-Impiomto et l., 1980). Furthermore these cells cn e esily induced to dopt well chrcterized physiologicl sttes modifying either the nture or the mount of the mitogens present in the

2 1456 cell medium (Alvrez et l., 1993). Like `in vivo' rt thyroid cells, FRTL-5 growth is lso inhiited y TGF-1 (Png et l., 1992). All of these chrcteristics mke FRTL-5 cell line good model to investigte the ction of TGF-1. Therefore, we studied the e ects of TGF-1 on FRTL-5 cells under vrious experimentl conditions such s resting cells, resting cells stimulted to proliferte y the ction of vrious mitogens, nd ctively proliferting cells. Results TGF-1 e ects on cell cycle relted genes in FRTL-5 proliferting cells TGF-1 inhiited the growth of ctively cycling FRTL-5 cells (Figure 1). TGF-1 rrested FRTL-5 cells in the G 0 /G 1 phse of the cell cycle s seen y ow cytometry (dt not shown). We estimted protein nd mrna levels of vrious cell cycle relted genes tht hve een shown to e ltered fter TGF1 tretment (Polyk, 1996). Mouse mcrophges express two mrna species of 3.8 nd 4.5 k (Mtsushime et l., 1991). However, in rt intestinl cells only the lrger 4.5 k is detected (Ko et l., 1995). We detected the two mrna species in our FRTL-5 cells. Cyclin D1 mrna levels were slightly decresed (Figure 2) wheres protein levels were mrkedly reduced (Figure 2). Cyclin D3 mrna levels (dt not shown) nd protein levels were not ected y TGF-1, while cyclin D2 could not e detected in these cells. In FRTL-5 cells cyclin E is expressed s group of nds with moleculr weight etween 46 nd 51 kd. Addition of TGF-1 to proliferting cells did not ect either cyclin E protein levels or the moility of the nds (Figure 2), n indirect re ection of its phosphoryltion (Dulic et l., 1992). Altogether, these dt suggest TGF-1 selective inhiition of expression in FRTL-5 c Figure 1 Inhiition of cell prolifertion y TGF-1. () Proliferting cells were treted with di erent doses of TGF-1 or vehicle for 2 dys. () Serum strved cells were stimulted with growing medium (5% NCS5H) or (c) TSHIGF-1 with or without TGF1. After 2 dys of incution, cell prolifertion ws mesured y the MTT ssy (see Mterils nd methods). Results represent the men of four di erent experiments mde y triplictes.e. *P50.05

3 cells. CDK4 mrna levels remined invrile fter TGF-1 ddition (Figure 2). On the other hnd p27 mrna levels were decresed oth t 24 nd 48 h fter the ddition of TGF-1 (Figure 2), lthough the protein levels were un ected (Figure 2). Proliferting FRTL-5 cells hve lmost undetectle levels of p15 mrna nd ddition of TGF-1 induced p15 mrna expression. Moreover, this induction seemed to increse with the length of the tretment (Figure 2). The mrna levels of c-myc, gene essentil for cell prolifertion, incresed fter TGF-1 ddition. Despite this, mrna levels of prothymosin lph (PTA), one of its puttive trget genes (Gutz et l., 1994), were initilly un ected nd ppered reduced with time, perhps re ecting the growth rrest of the cells, since PTA is not expressed in quiescent cells (Zlvide et l., 1992). We lso studied the complexes -p27 nd cyclin E-p27 y coimmunoprecipittion. To our surprise, TGF-1 treted cells hd comprle levels of ssocited with p27 to norml growing cells (Figure 3), despite of the fct tht totl protein levels were mrkedly reduced (Figure 2). When we immunoprecipitted the sme mount of protein from treted nd untreted cells ± more quntity of lyste from the TGF-1 treted cells ws required to mke even the immunoprecipitted levels ± we could detect thicker p27 nd in h cyclin D3 CDK4 cyclin E p15 c-myc PTA 18 S Figure 2 E ect of TGF-1 (2 ng/ml) on () mrna or () protein expression of severl cell cycle relted genes. Proliferting FRTL-5 cells were cultured in the sence or presence of TGF-1 nd totl mrna nd protein were extrcted t the indicted times. Filters were hyridized with di erent cdnas or ntiodies to severl cell cycle relted genes, 18 S rrna ws used s control to ensure equlity of loding mong lnes. Results shown were representtive of three independent experiments

4 1458 the TGF-1 treted lne (Figure 3). Since the mount of p27 ound to did not chnge (Figure 3), it seemed tht in the control cells we were immunoprecipitting some -CDK4/6 complexes free of p27 s well s complexes ound to p27; in contrst, in the TGF-1 treted cells we were immunoprecipitting minly p27--cdk4/6 complexes nd we could see igger nd. This result supports tht TGF-1 tretment gretly reduces D1-CDK4/6 complexes free of p27. Further support to this hypothesis cme when immunoprecipitting equl mounts of lystes ± tht mens di erent quntities of ± similr p27 nd ws otined (Figure 3c). We concluded tht the totl mount of p27 ound to ws not ected y TGF-1, indicting tht TGF-1 ction on FRTL-5 cells ected cyclin D1-CDK4/6 free of p27, ut not p27-ssocited cyclin D1-CDK4/6. On the other hnd, we found tht s result of TGF-1 tretment, the mount of p27 ound to cyclin E-CDK2 ws gretly incresed (Figure 4). In ccordnce with this, cyclin E-CDK2 kinse ctivity ws clerly reduced (Figure 4) nd prb ws hypophosphorylted in cells treted with TGF-1 (Figure 4c). TGF-1 regultion of FRTL-5 restimulted cells When resting FRTL-5 cells were induced to proliferte y growing medium (5% NCS plus 5H) the simultneous ddition of TGF-1 signi cntly reduced cell prolifertion (Figure 1). However when the mitogenic stimulus ws TSH plus IGF-1, TGF-1 hd no e ect (Figure 1c). Signi cntly, TGF-1 hd greter e ect decresing levels when the mitogenic stimulus used ws growing medium thn when TSHIGF-1 ws IP p27 w S kd w p27 S p27 IP c IP w w p27 w p27 kd S 51.6 S p27 Figure 3 E ect of 24 h tretment with TGF-1 on the G1 cyclin-cdk-p27 complexes in proliferting FRTL-5 cells. () 2 mg of totl cell lystes were immunoprecipitted with nti-p27 nd immunolotted ginst or p27. () Equl mounts of cyclin D1 were immunoprecipitted with nti- ntiody nd immunolotted ginst or p27. (c) 1 mg of totl cell lystes were immunoprecipitted with nti- ntiody nd immunolotted ginst p27. S=stright lyste loded s running control

5 used (Figure 5). At 8 h cyclin D3 mrna levels correlte well with mrna expression showing high induction in their protein levels fter the ddition of the two groups of mitogens (Figure 5) tht ws strongly reduced y TGF-1 on cells stimulted with growing medium, ut only slightly decresed in cells stimulted with TSHIGF-1. Similrly, cyclin E ws induced y mitogens nd this induction ws prevented when TGF-1 ws dded simultneously with growing medium, ut not in TSHIGF-1 treted cells (Figure 5). The expression of D-type cyclins ws relted to the phosphoryltion sttus of prb, good index of its ctivity. TSHIGF-1 tretment incresed prb degree of phosphoryltion compred to the resting cells (Figure 5c) nd this ws un ected y TGF-1 tretment. A similr increment in phosphoryltion ws seen fter growing medium ddition, ut, in this cse, TGF-1 ddition mintined prb in hypophosphorylted sttus (Figure 5c). The high levels of p27 mrna present in quiescent cells were strongly reduced fter TSHIGF-1 tretment nd remined decresed fter TGF-1 ddition (Figure 5). p27 protein levels were further reduced when TGF-1 ws dded (Figure 5). In contrst, p15 mrna levels were low in quiescent cells nd no p15 mrna ws detected fter TSHIGF-1 stimultion. After TGF-1 ddition, n increse in p15 mrna levels ws oserved (Figure 5). There ws diminution in c-myc mrna fter 24 h of TSHIGF-1 incution (Figure 5), re ecting tht c-myc induction y TSHIGF-1 occurs erlier (dt not shown). PTA mrna levels incresed fter mitogenic stimultion (Figure 5). Interestingly, reduction in PTA mrna levels ws oserved fter TGF-1 ddition, even in those cells tht were not rrested y the tretment, despite c-myc mrna levels eing incresed. TGF-1 e ects in quiescent cells We wnted to know the functionl e ect of TGF-1 on serum deprived FRTL-5 cells. We counted cells, with or without TGF-1, nd found decresed cell numer in TGF-1 treted cells (Figure 6). However, MTT ctivity incresed (dt not shown), indicting n ctivted metolism, perhps s result of poptosis (Truth nd Kessey, 1997). Therefore, we isolted solule cytoplsmic DNA nd found n internucleosoml ldder chrcteristic of poptosis fter 2 dys of tretment with TGF-1 (Figure 6). DNA frgmenttion ws con rmed y mens of TUNEL ssy. After 4 dys of tretment we could see very strong lel in treted cells compred with the controls (Figure 7). This ws ccompnied y chnges in cytoesqueleton structure re ecting the descried ctions of TGF-1on cell dhesion nd micro lment orgniztion (MssgueÂ, 1990). After the ddition of TGF-1, resting cells presented drmticlly incresed levels of p15 nd c-myc mrna (Figure 8). Cyclin D1 mrna nd protein levels, lredy low in resting cells, were further decresed y TGF-1 tretment (Figure 8 nd ). Cyclin E protein levels were unchnged (Figure 8) fter TGF-1 ddition nd p27 mrna nd protein levels were strongly reduced (Figure 8 nd ). There ws less p27 ssocited to cyclin E (Figure 8c) lthough this did not result in chnge of ctivity mesured in vitro y histone H1 phosphoryltion, or in vivo y prb phosphoryltion sttus (Figure 8d nd e). Discussion Physiopthologicl situtions cn chnge the size of the thyroid glnd. It increses fter goitrogen tretment or regresses fter goitrogen withdrwl. The size of the 1459 IP cyclin E IP cyclin E/ H1 kinse ssy w p27 S kd 51.6 H p c kd prb 118 Figure 4 Cyclin E-CDK2 complexes ctivity. () 2 mg of totl protein were immunoprecipitted nti-cyclin E nd immunolotted ginst p27. A thick nd of out 52 kd corresponding to the Ig hevy chin is seen in the immunoprecipitted lnes () Cyclin E-CDK2 complexes were ssyed for ssocited kinse ctivity using histone H1 s sustrte (c) 90mg of totl cell extrct were loded onto 7% SDS-Polycrilmide gel nd Western lot nti-prb ws done to see the di erent prb phosphorylted nds

6 1460 glnd is the result of precise lnce etween vrious stimuli. In generl, the mjor element tht controls thyroid growth in vivo is TSH. In vitro, optiml growth e ects of TSH require the presence of IGF-1 or Insulin, s comitogenic fctor (Dumont et l., 1992). In FRTL-5 resting cells, TSH nd IGF-1 ct s competence nd progression fctors respectively (Alvrez et l., 1993; Tkhshi et l., 1990). As in other epithelil cells, TGF-1 hs een reported to inhiit thyroid growth oth in vivo nd in vitro. In rts, it hs een shown tht there is n increse in locl expression of TGF-1 during goiter hyperplsi. This incresed expression is mintined through glnd involution fter goitrogen withdrwl, suggesting tht TGF-1 my contriute to the control of goiter size in response to di erent stimuli (Logn et l., 1994). Like in vivo, FRTL-5 growth is lso inhiited y TGF-1 (Png et l., 1992). In the present report, we hve studied the e ect of TGF-1 tretment on FRTL-5 thyroid cells re ecting vrious physiologicl situtions such s resting cells, resting cells stimulted to proliferte y the ction of vrious mitogens, nd ctively proliferting cells. TGF-1 induced poptosis in quiescent cells, rrested proliferting cells (Png et l., 1992; Morris et l., 1988) nd ws le to inhiit the growth of cells stimulted with growing medium ut not when stronger mitogenic stimulus, TSHIGF-1, ws used. These e ects on FRTL-5 could ccount for some of the chnges seen in the thyroid glnd fter the ddition or withdrwl of goitrogenic gents. Moreover, FRTL-5 cells provide n interesting model to study the mechnisms of ction of TGF-1. In ll situtions we found tht TGF-1 decresed p27 mrna nd levels nd, on the other hnd, incresed oth p15 nd c-myc mrna. TGF-1 t lest in prt inhiited growth in proliferting thyroid cells through mrked decrese in cyclin E-CDK2 kinse ctivity, ssessed in vitro using histone H1 s sustrte. In ccordnce with this, prb ws found to e underphosphorylted in TGF-1 treted cells. This inhiition of kinse ctivity seemed to e medited y p27 inding to the cyclin E-CDK2 complexes. However, no chnges were seen in cyclin E or p27 totl protein levels. p27 mrna levels diminished nd p27 protein did not chnge, perhps s result of di erent control of p27 trnscription nd trnsltion s previously descried (Pgno et l., 1995; reviewed in Sherr nd Roerts, 1995). It hs een proposed tht TGF-1 tretment induces the expression of p15 tht shifts p27 from -CDK4/6 to cyclin E-CDK2 complexes (Reynisdo ttir et l., 1995). However, despite n increse in p15 mrna levels, the mount of -ound p27 did not chnge in FRTL-5 cells. The tretment with TGF-1, even if cellulr p27 levels remined invrint, incresed p27 ound to cyclin E ut did not ect p27 ound to, ringing up the question of the origin of p27 ound to cyclin E. This cn e explined y the model TSH IGF-1 8h TSHIGF 1 NCS5H 24 hours cyclin D3 cyclin D3 cdk4 cyclin E p15 c-myc PTA c P Q TI TI NCS5H NCS5H prb 18 S Figure 5 E ect of TGF-1 tretment on () mrna nd () protein levels of severl cell cycle relted genes in quiescent FRTL-5 cells. Cells were cultured in low serum medium (0.5% NCS withou 5H), s descried in Mterils nd methods. At the indicted times, TSH (6 mui/ml) plus IGF-1 (30 ng/ml) or norml growing medium (5% NCS plus 5H) were dded in the sence or presence of TGF S rrna ws used s control to ensure equlity of loding mong lnes. (c) Western lot nlysis of prb phosphoryltion sttus in extrcts from proliferting (P), serum deprived (Q) nd restimulted cells with TSH plus IGF-1 (TI) or norml growing medium in the sence or presence of TGF-1

7 proposed in Figure 9. Proliferting cells hve prt of -CDK4/6 complexes ound to p27 nd prt free of it. Some p27 is not le to join - CDK4/6 either due to its phosphoryltion stte (Sherr nd Roerts, 1995) or ecuse it is ound to other/s sequestering proteins. After TGF-1 tretment (Figure 9), inctive p27 is ctivted y de-/phosphoryltion (Sherr nd Roerts, 1995) or shifted o the sustrte tht retined it, eing le to ind cyclin E-CDK2. Immunoprecipittion ginst p27 revels the sme 1461 p Mrker Figure 6 TGF-1 induces poptosis in quiescent FRTL-5 cells. () Serum nd hormonl deprived cells were treted with TGF-1 nd the numer of cells ws mesured t the indicted times using Coulter counter. () Cytoplsmic DNA from TGF-1 treted cells ws extrcted, loded into 1.5% grose gel nd stined with ethidium romide. After 48 h of tretment n poptotic ldder ppered in TGF-1 treted cells Figure 7 Morphologicl chnges nd poptosis induction on FRTL-5 quiescent cells treted with TGF-1 during 4 dys. (,) controls or (c,d) TGF-1 treted cells were xed nd processed for TUNEL ssy (,d). As control of cell morphology, cytoeskeleton polymerized ctin ws stined with TRITC-Phlloidine (,c). Cells were photogrphed t 2006mgni ction, with the sme exposure time, in Zeiss uorescence microscope

8 h p15 c IP cyclin E 48h w p27 kd 51.6 c-myc CDK4 PTA p s d IP cyclin E/ H1 kinse ssy Cyclin D1 Cyclin E H1 Cyclin D3 e kd prb 118 Figure 8 E ect of TGF-1 on quiescent cells. FRTL-5 quiescent cells were otined s descried in Mterils nd methods nd () mrna nd () protein levels of severl cell cycle relted genes were nlysed. (c) Cyclin E-CDK2-p27 ssocition ws studied nd (d) cyclin E-CDK2 ctivity ws mesured in vitro using histone H1 s sustrte. (e) In vivo prb phosphoryltion ws lso determined mount of ssocited in oth TGF-1 treted nd untreted cells, excluding the possiility of eing more p27 ound to the remining - CDK4/6 complexes in TGF-1 treted cells. The ction of TGF-1 seems directed t reducing p27-free cyclin D1 (Figure 9). Three possile mechnisms could explin this reduction. Firstly, direct e ect of TGF1 on the trnscription of, s proposed y others (Ko et l., 1994). Alterntively, the reduction on protein levels could e medited y the incresed levels of c-myc induced y TGF-1; it hs een lredy descried trnscriptionl inhiitory e ect of c-myc on gene (Philipp et l., 1994). However, it hs een reported tht TGF-1 inhiited c-myc trnscription in kertinocytes nd thyroid crcinom cells (Khosl et l., 1994; Alexndrow et l., 1995), in contrst with our results, perhps re ecting di erences in TGF-1 ction depending on the cell type studied. Finlly, the reduction of cyclin D1 could e explined y TGF-1 disrupting cyclin D1-CDK4/6 ssocition, leving free tht is rpidly degrded (Btes et l., 1994). Cyclin D1- CDK4/6 complexes cn e disrupted decresing CDK4 levels. We did not nd consistent chnges of CDK4 mrna expression fter TGF-1 tretment. We cnnot disregrd direct inhiition of CDK4 Figure 9 Model explining the results found in proliferting thyroid cells upon TGF-1 ction. Cyclin D1-CDK4/6-p27 complexes remin invrint while -CDK4/6 complex free of p27 dispper. Inctive p27 cn e ctivted y de- or phosphoryltion or y eing shifted o sequestering sustrte, llowing its union to cyclin E-CDK2 complexes. As result prb would remin hypophosphorylted

9 trnsltion ut not trnscription in TGF-1 treted cells (Ewen et l., 1995). On the other hnd, TGF-1- medited induction of p15 might led to its incresed ssocition with CDK4 nd disruption of cyclin D/ CDK4 complexes. We certinly found incresed p15 mrna levels fter TGF-1 tretment. Further experiments re now required to clrify this point. Quiescent FRTL-5 cells re strongly induced to proliferte fter the ddition of growing medium (5% NCS plus 5H) or codministrtion of higher doses of only two hormonl signls, TSH nd IGF-1, t low serum levels. TGF-1 ws le to inhiit the growth of cells stimulted with growing medium nd hormones ut not when TSHIGF-1, stronger mitogenic stimulus, ws used. This pprent prdox could e explined considering tht the mitogenic stimulus increses the levels of limiting fctor necessry for cell prolifertion. This is model tht llows etter chrcteriztion of the cell cycle regultory proteins tht could e ected y TGF-1 ction. We hve found tht oth growing medium nd TSHIGF-1 incresed the levels of, cyclin D3 nd cyclin E. TGF-1 hd greter e ect decresing the levels of the forementioned proteins with growing medium rther thn when TSHIGF-1 ws used, in greement with the ntiprolifertive e ect of TGF-1. Both in TSHIGF-1 nd growing medium treted cells, p27 ws diminished y mitogens nd interestingly ws further decresed y TGF-1. Finlly, TGF-1 tretment of resting FRTL-5 cells induced cler reduction of cell numer fter 4 dys of tretment. The reduction ws ccompnied y DNA frgmenttion s seen y electrophoresis nlysis nd TUNEL rection, de nitely indicting tht TGF-1 tretment during 4 dys induced poptosis of resting FRTL-5 cells. TGF-1 incresed c-myc mrna levels nd, therefore, c-myc could hve een mediting the poptotic e ect. Indeed, the increse in c-myc expression in growth fctor deprived cells is ccompnied y the rpid induction of poptosis (reviewed in Pckhm et l., 1995). FRTL-5 quiescent cells still hve detectle levels of cyclin E tht were not modi ed y TGF-1. However, cyclin E-CDK2 ctivity ws low, s shown y in vitro H1 phosphoryltion or in vivo prb hypophosphoryltion. On the other hnd, TGF-1 decresed p27 protein levels suggesting di erent regultion of p27 in quiescent nd proliferting cells. Most of the cell cycle regultory proteins conform n intricte network of mutul ctivtors nd inhiitors. The e ects of one memer of the pthwy cn e u ered or potentited y the others. Therefore, when TGF-1 cts on cell modifying the levels of one or severl cell cycle regultory proteins, the cellulr response will lso depend on the levels of the other cell cycle regultory proteins t the moment of ction. We hve found tht TGF-1 e ects on, c-myc nd p15 were lwys the sme despite the vriility of the cellulr responses. This nding indictes tht the forementioned regultory proteins could e key trgets of TGF-1 ction irrespective of the nl cellulr response. In summry, using FRTL-5 cells we developed model in which the TGF-1 ction on thyroid cells found in vrious physiologicl situtions such s resting cells, resting cells stimulted to proliferte y the ction of vrious mitogens, nd ctively proliferting cells cn e dissected. These e ects on FRTL-5 could ccount for some of the chnges seen in the thyroid glnd fter ddition or withdrwl of goitrogenic gents. Further studies re now required to estlish the importnce of TGF-1 in the control of thyroid size in vivo. Mterils nd methods Cell culture FRTL-5 cells were otined from the Europen Collection of Animl Cell Culture. Cells were grown in Coon's modi ed Hm's F-12 medium supplemented with 5% neworn clf serum (NCS; Gico BRL), 2 mm Glutmine (ICN Flow) nd ve hormone mixture (5H: 1 nm hydrocortisone, 5 mg/ml trnsferrin, 10 ng/ml somtosttin, 1 mui/ml TSH nd 10 mg/ml insulin; Sigm). To otin-serum strved FRTL-5, cells were wshed three times with PBS nd incuted in medium with 0.5% NCS nd without 5H for 2 dys. Quiescent cells were stimulted with TSH (6 mui/ml) nd IGF-1 (30 ng/ml). Recominnt humn TGF-1 (Amershm) ws diluted in 0.4 mm HCl, 1% BSA. TGF-1 exerted n inhiitory ction on FRTL-5 prolifertion t severl doses (Figure 1). TGF-1 doseof 0.5 ng/ml ws not le to mintin the inhiition fter 4 dys of tretment (dt not shown). Bsed on these preliminry results, we used dose of 2 ng/ml of TGF-1 in the susequent studies. Prolifertion ssys For prolifertion ssys, cells per well were seeded in 24-well pltes (Corning). After 3 dys, the medium ws chnged nd cells were treted with TGF-1 (2ng/ml)or vehicle. In serum-strved experiments, cells were chnged to low serum medium (0.5% NCS without 5H) for 2 dys prior to the tretment. Cell viility ws ssessed y the MTT ssy (Green et l., 1984). In rief, MTT ws dded to the medium t concentrtion of 0.5 mg/ml nd cells were mintined t 378C, 5% CO 2. After 4 h, 1 ml of 10% SDS, 0.01 M HCl ws dded followed y overnight incution. Asornce ws mesured t 550 nm in spectrophotometer. There ws liner reltionship etween cell numer nd MTT sornce etween 5000 nd cells (dt not shown). Alterntively, cells were counted in Coulter counter. Northern lot nlysis Totl RNA ws isolted y the gunidinium-thiocyntephenol-chloroform method (Chomczynski nd Scchi, 1987). 20 mg of totl RNA were run in 1.2% formldehyde grose gel, stined with ethidium romide nd trnsferred y cpillrity. cdna proes were lelled with 32 P-dCTP (3000 Ci/mmol) (Amershm) using rndom primed kit (New Englnd Biols). Plsmids contining mouse p15 nd mouse p27 cdnas were otined from Chrles J Sherr nd Jon Mssgue respectively. Mouse c-myc cdna (Stnton et l., 1983), humn prothymosin lph cdna (Goodll et l., 1986), humn nd cdk4 cdnas (Hinds et l., 1992) nd humn cyclin D3 cdna (Motokur et l., 1992) were lso used s proes. An oligonucleotide for the 18S rrna (ACGGTATCT- GATCGTCTTCGAACC) ws used s n internl control to ensure equlity of loding mong lnes (Soto et l., 1995). Immunolotting nd immunoprecipittion Cells were wshed twice with PBS nd incuted for 30 min t 48C) in n ice-cold lysis u er (50 mm HEPES 1463

10 1464 ph 7.5, 150 mm NCl, 10% glycerol, 1% Triton X-100, 5mM EGTA, 1.5 mm MgCl 2, 20 mm N 4 P 2 O 7 20 mm N 3 VO 4, 50 mg/ml protinin nd 4 mm PMSF). Lystes were collected with n scrpper, centrifuged ( g, 15 min 48C) nd superntnts were kept t 7808C. Sixty mg of totl proteins were resolved y SDS ± PAGE nd electrotrnsferred onto nitrocellulose memrne (Schleicher & Schuell). Immunodetection ws crried out using chemioluminiscent system (Tropix) following mnufcturer's instructions. Primry ntiodies ginst p27,, cyclin D3 nd cyclin E (Snt Cruz Biotechnology) were used t 1 : 1000 dilution. For immunoprecipittion ssys, cells were lysed s descried ove nd the indicted mounts of totl protein were incuted for 3 h t 48C with the primry ntiody. Then, ddition of 30 ml of protein G-Sephrose (Phrmci Biotech) per smple ws followed y 45 min incution t 48C. After centrifugtion, the pellets were wshed ve times with HNTG u er (20 mm HEPES ph 7.5, 150 mm NCl, 10% glycerol, 0.1% Triton X-100) nd resuspended in 30 ml of protein smple u er. Histone H1 kinse sssy Cells lysis nd immunoprecipittion were performed s descried ove. After wshing in HNTG u er, the pellets were resuspended in 40 ml of kinse u er (20 mm HEPES ph 7.4, 5 mm N 3 VO 4, 10 mm MgCl 2, 25 mm EGTA, 10 mm -glycerophosphte, 20 mm ATP) contining 10 mci of [g- 32 P]ATP (3000 Ci/mmol) (Amershm) nd 1 mg of histone H1 (Boehringer Mnnheim). Smples were incuted t 378C for 30 min nd oiled in protein smple u er. Proteins were resolved on 12% SDS ± PAGE, trnsferred nd visulized y utordiogrphy. Anlysis of DNA frgmenttion TGF-1 ws dded to serum-deprived FRTL-5 cells ( ) t the indicted times. Anlysis of DNA frgmenttion ws performed s previously descried (Trent et l., 1996) with minor modi ctions. Brie y, the cells were hrvested nd wshed twice with PBS. The pellets were resuspended in 0.4 ml of lysis u er (10 mm Tris ph 8.0, 1 mm EDTA ph 8.0, 0.2% Triton X-100) nd incuted for 30 min on ice. After centrifugtion t g for 10 min, DNA from the superntnts ws precipitted overnight t 7208C y dding of 0.5 volumes of isopropnol nd NOH to nl concentrtion of 0.5 M. Smples were centrifuged nd pellets were resuspended in TE u er nd incuted 1 h t 378C with RNAse A (100 mg/ml) nd 2 h t 508C with proteinse K (300 mg/ml). Smples were electrophoresed through 1.5% grose gel in TAE u er t 65 V. DNA nds were visulized y stining with ethidium romide. TUNEL ssy DNA frgmenttion ws ssyed y the TUNEL rection (Gvrieli et l., 1992). Brie y, cells were ttched to the coverslide with 4% prformldehyde nd permeilized in HEPES u er (1 M HEPES ph 7.5, 300 mm sucrose, 50 mm NCl, 3 mm MgC 2 ) with 0.5% Triton X-100 during 4 min on ice. After wshing with PBS, the TUNEL rection ws performed with the `In situ Cell Deth Detection Kit' (Boehringer Mnnheim) following the mnufcturer's instructions. As control of cellulr morphology TRITC-Phlloidin (2 mg/ml) ws dded to the TUNEL rection. Fluorescence ws oserved t 2006 mgni ction with Zeiss uoresence microscope. Sttisticl nlysis Results re expressed s percentge with respect to the control. A non-prmetric Mnn ± Whitney test ws done to perform sttisticl nlysis of the dt. Acknowledgements We re grteful to Jon Mssgue nd Chrles J Sherr for providing us with p27 nd p15 plsmids respectively. We lso thnk Jvier Leo n nd Igncio Plmero for helpful discussions nd comments on the mnuscript. This work ws prtly supported y Xunt de Glici grnt to CAV (XUGA 30102A96) nd FISS grnts (Fondo de Investigciones Snitris de l Seguridd Socil) nd Consellerõ de Snidde e Servicios Sociis, Direccio n Xerl de Sude Pu lic, Progrm de Screening de Cncer de Mm, Xunt de Glici to FD. 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