Pro. Natl. Aad. St. USA Vol. 76, No. 4, pp. 222-226, April 1979 Medial Sienes Thyrotropin is not a growth fator for human thyroid ells in ulture (pituitary trophi hormones/goiter/ell differentiation) BNGT WSTRMARK*, F. ANDRS KARLSSON, AND OLOV WALINDR The Wallenberg Laboratory and the Department of Internal Mediine and Clinial Researh II, University Hospital, Uppsala, Sweden Communiated by Keith R. Porter, January 18, 1979 ABSTRACT Thyroid ells, obtained from both normal human tissue and benign nodular goiter, were ultured and maintained in vitro in 4-18 passages. Cultures with onfluent ells aumulated yli AMP (1-15 times the basal amount) upon addition of bovine thyrotropin (1 milliunits/ml), indiating that the ells in ulture maintained a thyrotropin-sensitive adenylate ylase system. Addition of high doses of thyrotropin also indued a harateristi and reversible hange in the morphology of the ells. The effet of thyrotropin on ell growth was studied in shortand long-term eperiments. Thyrotropin redued [3lljthymidine inorporation in a dose-dependent fashion in all ultures of thyroid ells. The maimal inhibition over a 24-hr period was about 5%. The thyroid ells were notably sensitive, and the half-maimal effet ourred at about 1 milliunits of thyrotropin per ml. In ontrast, the hormone had no effet on [3Hthymidine inorporation into human glial ells. Low doses of thyrotropin also had no effet on human fibroblasts and, at high doses, a stimulation of [3H]thymidine inorporation was seen. Thyroid ell ultures grown in the presene of 1 milliunits of thyrotropin per ml for 7-14 days had a slower growth rate and 24-36% lower ell numbers at saturation density than ontrol dishes, indiating that the hormone also had a long-term effet on ell proliferation. The data agree with in vitro studies by others of the effets of ortiotropin and lutropin on target ells and suggest that in vivo the primary ation of pituitary trophi hormones on endorine tissues is not stimulation of growth. Isolated thyroid ells from various speies, inluding human beings, an be maintained in ulture for a limited period of time (1-1). The addition of thyrotropin (TSH) to the ultures at the time of eplantation makes the ells organize into folliular strutures (3-7) that maintain the apaity to onentrate iodide and synthesize iodinated thyroglobulin (7, 11). It is generally assumed that an inreased yli AMP level has an important role in the transmission of the hormonal effets sine dibutyryl yli AMP and phosphodiesterase inhibitor an mimi ertain TSH ations on ells in vitro (5-7) and TSH ativates a membrane-bound adenylate ylase in thyroid ells (reviewed in ref. 12) İn several ell systems the development of differentiated funtions is aompanied by an inhibition of ell multipliation (reviewed in ref. 13). TSH is both onsidered important for the epression of differentiated funtions of the thyroid and generally believed to be a trophi fator for the thyroid in vivo. The objet of the present investigation was to analyze the effet of TSH on thyroid ell division in vitro by using human thyroid ell lines. The results demonstrate that TSH is not a growth fator for thyroid ells in vitro and have impliations for the understanding of thyroid growth in vivo. The results suggest that in the development of goiter the growth stimulus is not provided by TSH. MATRIAL AND MTHODS Materials. Bovine TSH (3 units/mg) was purhased from Ferring AB (Malm6, Sweden). Human TSH (2.5 units/mg) was kindly provided by AB Pharmaia (Uppsala, Sweden). The thyrotropin solutions were prepared by dissolving the hormone in.2 M phosphate buffer/. 15 M NaCl at ph 7.4, ontaining 1 mg of bovine serum albumin per ml, and dialyzing against the same buffer at 4 C overnight. Collagenase (153 units/mg) was purhased from Worthington; 3-isobutyl-1-methylanthine from Aldrih (Gillingham, U.K.); [3H]thymidine (2 Ci/mmol, 1 Ci = 3.7 X 11 Bq) from Radiohemial Centre (Amersham, U.K.); N6,2'-dibutyryl yli AMP from Sigma; and human serum albumin from AB Kabi (Stokholm, Sweden). A radioimmunoassay kit for determinations of yli AMP was obtained from Shwarz/ Mann. Cell Cultures. Thyroid ell lines were initiated from two nonfuntioning thyroid adenomas (lines designated HTh 1 and HTh 11), one funtioning adenoma (HTh 31), and in one ase from nonpathologial tissue surrounding a funtioning adenoma (HtTh 14). The tissue was fragmented with a salpel, suspended in phosphate-buffered saline ontaining 1 mg of ollagenase per ml, and inubated in a 37 C roking waterbath for 6 min. Cells were further liberated from the fragments by pipetting. Large fragments were allowed to sediment and the supernatant was deanted and entrifuged (2 X g for 1 min). The pellets were suspended in agle's minimum essential medium (14) supplemented with 1% fetal alf serum, 1 units of peniillin, 1.25,gg of amphoteriin B, and 5,ug of streptomyin per ml. The ells were inubated in 5-mm Nun petri dishes (Nun, Roskilde, Denmark). About 2 dishes were initiated from 1 g of wet weight tissue. The ultures were inubated at 37 C in a humidified atmosphere ontaining 5% CO2. Primary onfluent monolayers were usually formed within 7-14 days. The ultures were then subultivated at 1:2 split ratio. Cells were detahed after inubation in phosphate-buffered saline with.2% DTA for 5 min followed by a film of phosphate-buffered saline with.25% trypsin for about 1 min. Cultures of normal human skin fibroblasts and human glial ells were initiated and maintained as desribed (15, 16). Inorporation of [3HlThymidine. Cells were detahed and suspended in minimum essential medium ontaining 1% fetal alf serum and seeded into 35-mm Nun dishes. One 5-mm dish with a onfluent monolayer was split into eight 35-mm dishes. After 24 hr of inubation, medium was hanged to thymidine- and serum-free Ham's nutrient miture F-1 (17). After 24 hr of serum starvation, medium was hanged to F-1 ontaining 1 mg of human serum albumin per ml; fetal alf serum and TSH were added as speified below. The ultures The publiation osts of this artile were defrayed in part by page harge payment. This artile must therefore be hereby marked "advertisement" in aordane with 18 U. S. C. 1734 solely to indiate * Present address: Department of Moleular, Cellular and Develop- Abbreviation: TSH, thyrotropin. this fat. mental Biology, University of Colorado, Boulder, CO 839. 222
Medial Sienes: Westermark et al. Pro. Nati. Aad. S. USA 76 (1979) 223 p 49 4!w FIG. 1. (Left) Appearane of human thyroid ells obtained from benign nodular goiter (HTh 1). (Right) Same ells after treatment for 6 min with bovine TSH (1 milliunits/ml). were preinubated for 18 hr. Tritiated thymidine was then added to a final onentration of.2 AiCi/ml. After 24 hr of inubation, the ultures were harvested and radioativity inorporated into trihloroaeti aid-preipitable material was measured as desribed (18). Measurement of Cyli AMP. The proedure was essentially that desribed by Rapoport (9) for studies of maimal yli AMP prodution in dog thyroid ell ultures. Dupliate onfluent ell ultures were inubated with 1 milliunits of bovine TSH per ml and.5 mm 3-isobutyl-1-methylanthine and ontrol dishes with.5 mm isobutylmethylanthine for 6 min at 37C. Cyli AMP was measured by use of a radioimmunoassay kit based on the method of Steiner et al. (19). Determination of Cell Number. Cells were detahed in.2% DTA/.25% trypsin and ounted in an eletroni partile ounter (Cellosope, AB Ljungberg, Stokholm, Sweden). Protein Determination. Cells were lysed with.3 M NaOH and protein ontent was determined by the method of Lowry et al. (2). RSULTS Culture and Morphology of Human Thyroid Cells. In the present investigation, we tried to initiate ell lines from 21 thyroid biopsies of nonmalignant tissue. Cell growth ourred in all ases. However, the majority of ell ultures gave rise to mied populations of epithelial ells and fibroblasts. In four instanes ell lines free of visible fibroblast ontamination were initiated. The ells grew as monolayer ultures whih initially ould be passaged one to two times a week (Fig. 1). After several Table 1. passages, growth rates typially delined until no inrease in ell number was deteted (see Table 1). Addition of bovine TSH (1-1 milliunits/ml) to the ulture medium had a marked effet on ell morphology. After a few hours' treatment of sparse and onfluent dishes with bovine TSH, the ells beame arborized (Fig. 1 right). A similar morphologial response was also obtained by addition of.5 mm dibutyryl yli AMP. Upon prolonged treatment with TSH, the ell pattern gradually reversed to the original. After 24 hr of inubation, very few arborized ells ould be deteted. At this state renewed hallenge with fresh TSH was without any influene on the appearane of the ells. TSH-Sensitive Cyli AMP Prodution in Human'Thyroid Cell Cultures. Basal intraellular levels of yli AMP in onfluent human thyroid ell ultures were 8-14 pmol of yli AMP per mg of ell protein. The hallenge of 1 milliunits of bovine TSH per ml for 6 min aused a 1- to 15-fold inrease in intraellular ontent of yli AMP in thyroid ell ultures (Table 1). The basal level and the degree of TSH stimulation orrespond to those found by Rapoport in studies of dog thyroid ell ultures (9) under similar eperimental onditions. ffet of TSH on [3H]Thymidine Inorporation and Cell Density in Thyroid Cultures. Addition of bovine or human TSH to thyroid ells ultured in the presene or absene of 1% fetal alf serum aused a dose-dependent redution in [3H]- thymidine inorporation in all thyroid ell lines studied (Fig. 2). The effet was readily detetable in ultures kept in 1% fetal alf serum. The presene of serum was, however, not an absolute requirement sine the inhibitory effet of TSH was notied and showed a similar dose-response relationship in serum-free ffet of TSH on thyroid ell ultures Cyli AMP, Life span, pmol/mg ell protein Cell line Tissue origin passages.5 mm MIX.5 mm MIX + TSH HTh 1 Nonfuntioning nodule 15 14 143 HTh 11 Nonfuntioning nodule 6 12 51 HTh 14 Normal tissue surrounding a funtioning nodule 4 ND ND HTh 31 Funtioning nodule 18 8 1173 MIX, methylisobutylanthine; ND, not determined.
224 Medial Sienes: Westermark et al. Pro. Natl. Aad. Si. USA 76 (1979) 8' HTh 4 A HTh II B -f HTh 4 e 16 6' I- 4'. 2 2a o S- 8 HTh 14 C HTh 31 D Human TSH, milliunits/ml C 4-2 1-. I~'.1.1 1 1 1.1.1 1 1 1 FIG. 2. ffet of bovine TSH (A-D) and human TSH () on [3H]thymidine inorporation (mean of dupliate dishes) into aidpreipitable material of human thyroid ells in ulture. TSH was added in doses indiated, alone () or along with 1% fetal alf serum (), to ultures starved for serum for 18 hr. [3H]Thymidine inorporation during the following 24-hr was then measured. ultures also. The sensitivity of different thyroid ultures to the inhibitory ativity of TSH of bovine or human origin was similar, with a half-maimal effet ourring at 1 mirounits/ml (Fig. 3). With inreasing TSH onentrations the inhibitory effet leveled off. The maimal redution in [3H]- thymidine inorporation obtained was about 5%. The following eperiment was performed to see whether the TSH-mediated inhibition of [3H]thymidine inorporation ould be overome by an inrease in serum onentration. Serumstarved ultures were fed with media ontaining different onentrations of fetal alf serum (1-3%) with or without 1 milliunits of bovine TSH per ml and the inorporation of 1 2 Fetal alf serum, % FIG. 4. Influene of inreasing onentrations of fetal alf serum on the effet of a fied amount of bovine TSH on [3H]thymidine inorporation into aid-preipitable material of human thyroid ells in ulture (line HTh 1). Fetal alf serum was added in onentrations indiated, alone () or along with 1 milliunits of bovine TSH per ml (). [3H]Thymidine inorporated between 18 and 42 hr after addition of TSH was then measured. [3H]thymidine was measured as above. As shown in Fig. 4, an inhibitory effet of TSH was notied at all serum onentrations tested. The relative inhibition was most pronouned at 1% fetal alf serum. 6 5' 4' R ON. 41), 3 2- 'a M_).1.1 1 1 1 FIG. 3. Dose-response urve for TSH inhibition of [3H]thymidine inorporation into human thyroid ells. Data from eperiments shown in Fig. 2 A-D were epressed as % [3H]thymidine inorporation (mean and SM) as a funtion of TSH onentration, taking the inorporation in ultures fed with 1% fetal alf serum alone as 1%. 1 1 3 6 9 15 Time, days FIG. 5. Long-term effet of TSH on thyroid ell growth. Sparse ultures (HTh 31) were plated and ultured in F-1 medium ontaining 1% fetal alf serum with or without human TSH (1 milliunits/ml) and medium renewed three times a week. Cell numbers were determined for dupliate dishes.
Medial Sienes: Westermark et al. Pro. Natl. Aad. Si. USA 76 (1979) 225 B A fn 4. CC 3. 2) 'a 2 Ī 1- aa.1.1 1 1.1.1 i 1 1 FIG. 6. ffet of bovine TSH on [3H]thymidine inorporation into aid-preipitable material of (A) human fibroblasts and (B) human glial ells in ulture. Bovine TSH was added in doses indiated, alone () or along with fetal alf serum (@) to ultures starved for serum for 18 hr. [3H]Thymidine inorporation during the following 24 hr was then measured. The long-term effet of TSH on thyroid ell growth in vitro was also investigated. These eperiments were arried out in medium with 1% fetal alf serum. Cultures kept in the presene of bovine TSH (1 milliunits/ml) had a slower growth rate throughout the period of growth to saturation than ultures kept in the absene of TSH. Consequently, the final saturation density was lower in TSH-treated ultures-35% lower as shown in Fig. 5 and 24% lower in a parallel set of eperiments (data not shown). Note that these figures are lose to the degree of lowered [3H]thymidine inorporation found in short-term ultures ultured in 1% fetal alf serum (Fig. 4). ffet of TSH on [3HlThymidine Inorporation in Human Fibroblasts and Glia Cell Cultures. [3H]Thymidine inorporation in human fibroblasts and human glia ells showed an opposite response to TSH ompared to thyroid ells. At low levels of TSH no effet on human fibroblasts ould be deteted, but at higher onentration (1-1 milliunits/ml), stimulation of [3H]thymidine inorporation was found (Fig. 6). Under serum-free onditions the fibroblast ultures showed an "'-2-fold inrease. When bovine TSH was added along with 1% fetal alf serum, a 9-fold inrease with the highest hormone onentration, 1 milliunits/ml, was determined. Human glia ell ultures were not affeted by bovine TSH when [3H]thymidine inorporation was studied under idential eperimental onditions (Fig. 5). Thus, the markedly different results with fibroblasts and glial ells indiate that thyroid ell ultures have a unique sensitivity to an inhibitory ativity of TSH on [3H]thymidine inorporation. DISCUSSION In the present study human thyroid ell ultures, apparently free of fibroblast-like ells, responded with a marked elevation in yli AMP after addition of high doses of bovine TSH. This indiates the presene of a TSH-responsive adenylate ylase system in the ell ultures. The amounts of yli AMP formed and the magnitude of the inrease were similar to those reported for dog thyroid ells in tissue ulture (9). In addition, the human ells underwent the same morphologial alteration after TSH addition as was desribed for dog thyroid ells (9). TSH inhibited the inorporation of [3H]thymidine into all thyroid ell lines, whih were derived from nonpathologial and funtional or nonfuntional nodular tissues. In ontrast, TSH had no effet on [3H]thymidine inorporation in glial ell ultures; when tested in fibroblast ultures, TSH was without effet at low onentrations, whereas at high onentrations a stimulation of the inorporation of [3H]thymidine was found. It is likely that the stimulation seen with high doses of TSH in fibroblast ultures was aused by growth fators previously found to ontaminate ertain TSH preparations (see ref. 21). These findings and the marked sensitivity of thyroid ell ultures to preparations of both bovine and human TSH, with notieable inhibition at doses within the physiologial range (.1 milliunit/ml) and half-maimal inhibition at one order of magnitude higher onentration, strongly suggest that this phenomenon reflets a speifi interation between TSH and the TSH reeptor of the thyroid ell. Culture of human thyroid ells has been desribed in two previous reports. In one study human thyroid ells were maintained in ulture for up to 6 days and thyroid hormone prodution was measured (1). No data on TSH effets on ell growth were given. In another study (8), the effet of TSH on ultured human, dog, and bovine thyroid ells was reported. Nononfluent thyroid ells (speies origin not given) grew faster to saturation density in the presene of 1 milliunits of TSH per ml than the ontrol ells. However, after 6 days in ulture the same terminal ell density was reahed in both instanes. Further, bovine TSH (1 milliunits/ml) was reported to stimulate the inorporation of [3H]thymidine in a linear fashion during a 6-hr inubation. This study thus reported observations that differ from those found by us. The reason for this disrepany is not lear. However, differenes in the homogeneity of the ell populations studied and possible impurities in the TSH preparations may be of importane.
226 Medial Sienes: Westermark et al. The lak of stimulation of TSH on thyroid ell multipliation in vitro found in the present study prompts a disussion of data onerning the effets of TSH on thyroid growth in vivo. TSH injetions give rise to a wide range of morphologial responses (see review, ref. 12), the earliest refleting an inreased thyroid seretion. After 2-4 hr an inreased ell height, suggestive of ell hypertrophy, an be observed. Prolonged administration of TSH for up to 6 days leads to an inrease in thyroid weight with proliferation of apillaries preeding an inrease of the parenhyma (22, 23). In rat or mouse thyroids hronially stimulated by TSH a 3-fold inrease of the total ell number, estimated by DNA determinations, has been reported (24-26). The labeling inde was higher in endothelial ells and fibroblasts than in follile ells (27). Thus, it is lear that the inreased growth of the thyroid gland aused by prolonged TSH administration an be attributed only to a minor degree to an atual development of new follile ells. Moreover, in the latter studies (24-27) animals were used that had been pretreated with iodine-bloking agents to depress thyroid hormone prodution and inrease endogenous TSH seretion. Suh treatment makes an interpretation of the pure TSH effets diffiult. Finally, TSH has no effet on thyroid ell division, but auses marked ellular hypertrophy when infused into dwarf mie over a 3-week period (28). Thus, in our opinion data supporting a view that TSH itself is a growth fator for thyroid epithelial ells in vivo are not onvining. It is oneivable that in the development of goiter the primary stimulus for thyroid growth is iodine defiieny or disturbed thyroid metabolism and-not TSH. The present results should be ompared to observations on the interation between other pituitary trophi hormones and their respetive target ells in ulture. Thus, lutropin has been reported to suppress the mitoti rate in ultured luteal ells (29), whereas differentiated luteal ell funtions are indued and maintained. An antimitoti effet of ortiotropin on rat adrenoortial (3) or rat adrenal tumor ells (31-33) in ulture has reently been doumented. All three hormones (ortiotropin, lutropin, and TSH) ativate membrane-bound adenylate ylase and thereby stimulate the hormone prodution of their respetive target ells. Taken together, the different studies support the onept that ell multipliation and epression of differentiated funtions may be antagonisti. While it is generally believed that TSH has a trophi ation in vivo, onlusive eperimental evidene supporting this onept is laking. The present study shows that TSH does not stimulate growth of human thyroid ells in ulture. It is suggested that TSH might mediate a proliferation response in vivo via the indution of speifi growth fators. The eellent assistane of Majstin Wiik-Lundberg and Annika Magnusson is gratefully aknowledged. This work was supported by the Swedish Medial Researh Counil (Projet 4996), the Swedish Caner Soiety (Projet 55), and a grant from the Harald and Greta Jeanson Foundation. Pro. Natl. Aad. Si. USA 76 (1979) 1. Pastan, I. (1961) ndorinology 68,924-931. 2. Tong, W., Kerkof, P. & Chaikoff, I. L. (1962) Biohim. Biophys. Ata 6, 1-19. 3. Kerkof, P., Long, P. J. & Chaikoff, I. L. (1964) ndorinology 74, 17-179. 4. Dikson, J. A. (1966) ndorinology 79,721-731. 5. Fayet, G., Paheo, H. & Tiier, R. (197) Bull. So. Chim. Biol. 52,299-38. 6. Knopp, J., Stol, V. & Tong, W. (197) J. Biol. Chem. 345, 443-448. 7. Lissitzky, S., Fayet, G., Giraud, A., Verrier, B. & Torresani, J. (1971) ur. J. Biohem. 24,88-99. 8. Winand, R. J. & Kohn, L. D. (1975) J. Biol. Chem. 25,6534-654. 9. Rapoport, B. 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