Exp. Clin. Endocrinol. 100 (1992) 57-61
Experimental and Clinical Endocrinology © 1992 Johann Ambrosius Barth
Thyrotropin Receptor and Growth of Thyroid Carcinomas M. Broecker' and M. Derwahi
Key words: TSH receptor - Thyroid carcinomas - Growth - Adenylate cyclase
Zusammenfassung: Aufgrund klinischer Erfahrungen wird allgemein angenommen, daß Thyreotropin (TSH) das Wachstum differenzierter Schilddrüsencarcinome stimuliert. Nicht alle klinischen Studien konnten jedoch diese Annahme bestätigen.
Ebenfalls widersprüchlich sind In-vitro- und In-vivo-
Untersuchungen an Transplantaten menschlicher Schilddrüsencarcinome, die entweder einen stimulierenden oder einen hem-
menden oder keinen TSH-Effekt auf das Tumorwachstum nachwiesen. Als gesichert gilt, daß der TSH-Rezeptor in differenzierten Schilddrüsencarcinomen exprimiert wird. Um auf molekularer Ebene die Bedeutung von TSH und seines Rezeptors an Zeilmodellen studieren zu können, etablierten wir zwei permanente Schilddrüsencarcinomzellinien. Durch Transfektion der humanen TSH-Rezeptor-cDNA in FRTL-5-Schilddrüsenzellen entstand die FTSHr-Tumorzellinie, die in vitro und
Thyrotropin (TSH) is widely believed to influence
als Transplantat auf Nacktmäusen TSH-unabhängig wächst, wichtige differenzierte Funktionen (Thyreoglobulinsynthese, Jod-Aufnahme) verloren hat, aber weiterhin ein funktionsfähiges TSH-Rezeptor-Adenylat-Zyklase-System besitzt. Die zweite Tumorzellinie HTC-TSHr wurde durch Transfektion der
humanen TSH-Rezeptor-cDNA in menschliche Schilddrüsencarcinomzellen, die keinen TSH-Rezeptor mehr exprimieren, geschaffen. Diese Zellinie reexprimiert den TSH-Rezeptor, der an das zelleigene Adenylat-Zyklase-System gekoppelt ist. Überraschenderweise führt eine TSH-Stimulation dieser Zellen zu einer Hemmung des Zellwachstums. Beide Tumorzellinien
belegen, daß der alleinige Nachweis einer TSH-RezeptormRNA-Expression, einer spezifischen TSH-Bindung oder einer TSH-abhängigen zyklo-AMP-Synthese nicht ausreichen, eine wachstumsstimulierende TSH-Wirkung abzuleiten. Ferner zeigt sich, daß TSH auch das Wachstum menschlicher Schilddrüsencarcinomzellen hemmen kann.
growth of thyroid carcinomas (Mazzaferri, 1981). This
expressed in differentiated carcinomas, albeit at a lower level than compared to normal thyroid tissue (Brabant et
view is supported by several clinical studies which
al., 1991;Ohtaetal., 1991).
revealed a positive therapeutic effect of a TSH-suppres-
To further elucidate the role of TSH and its receptor in thyroid carcinomas we established two in vitro models, the FTSHr cell line (Derwahl et al., 1992a) and the HTC-TSHr cell line (Derwahl et al., 1992 b).
sive treatment after ablative therapy in patients with thyroid carcinomas (Crile, 1955; Clark, 1981). However, other investigators were unable to confirm these results (Cady, 1983). Various in vitro studies performed during the last two
decades did not resolve this issue. In comparison with normal thyroid tissue TSH binding andlor TSH-stimu-
Materials and Methods
lated cAMP accumulation were found either to be identi-
Cell cultures
cal or increased or decreased in differentiated thyroid carcinomas (Crile, 1966; Schorr et al., 1972; Takahashi et al., 1978; Sand et al., 1976; Abe et al., 1977; Clark et al., 1978; Saltiel et al., 1981). However, it has been demonstrated recently that the TSH receptor mRNA is 1
Part of doctoral thesis at the University of Bochum
Monolayer cultures of cell lines were grown in Coon's modi-
fied Ham F12 medium supplemented with 10% fetal calf serum (HTC cells) or 5% calf serum (FRTL-5 cells) and six hormones or growth factors [glycyl-histidyl-lysine, 10 ng/ml; insulin, 10 sg/mI; somatostatin, 10 ng/ml; transferrin, 5 tg/ml; hydrocortisone, 3.2 ng/mIl with 10 mU/ml (H6-medium) or without bovine TSH (H 5-medium), 100 UI penicillinlml and 100 sg streptomycin/mI in 25 cm2 plastic culture flasks (Corn-
Downloaded by: University of Pittsburgh. Copyrighted material.
Laboratories of Endocrinology, University Clinic of Internal Medicine Bergmannsheil, Bochum, Germany
Exp. Clin. Endocrinol. loo (1992) 1/2
58
ing; New York, NY). Cells were passaged by enzymatic dissociation with trypsin EDTA (Seromed, Biochrom, Berlin,
[3H] thymidine incorporation
Germany).
For studies of [3H] thymidine incorporation the cells were cultured in 96 well plates in H 5 medium supplemented with the indicated concentrations of TSH or dibutyryl-cAMP or forskolin for 48 h. Then cells were removed from plates by treatment with trypsin and measured in a j3 scintillation
The human TSH receptor cDNA (hTSHR-cDNA) inserted into the Eco RI sites of the eukaryotic expression plasmid pSV2-NEO-ECE (kindly provided by Dr. B. Rapoport, San Francisco) was transfected into FRTL-5 cells and HTC cells using a modified lipofection method (Felgner et al., 1987). Briefly, plasmid DNA was mixed with cationic liposomes (10 sl per .tg DNA) and the formed DNA/liposome complex applied to a 100 mm culture dish. The monolayer cultures were incubated for 4 hours and then regular culture medium was added. 4-5 days after transfection 400 sg/ml Geneticin (0418) was added to the culture medium to select 1
transfected cells. After 4-5 weeks when non-transfected cells had died, cells were passaged and surviving clones were pooled. Transformed cells of three independent transfections were further subcloned by limiting dilution. As negative control, cells were transfected with the plasmid pSV 2-NEO-ECE without the hTSHR-cDNA.
Radiolabeled TSH binding
FTSHr cells were grown in 24 well plates to confluence. Binding studies were performed in Hank's modified buffer without NaCl (Tramontano et al., 1986). To maintain isotonicity 280 mM sucrose was added. Highly purified '25I]bTSH was a generous gift of Dr. Herzog (Henning Ber-
lin, Germany). Cells were incubated for 2 h at 37 °C with ['25IIbTSH and the indicated amount of unlabeled TSH (Sigma). At the end of the incubation cells were rinsed three times with the same buffer (4C) and solubilized with I N NaOH. Radioactivity was measured in a u-counter. Nonspecific '25I}bTSH was determined in presence of 10_6 M unlabeled TSH, and this value subtracted from total binding. The data are expressed as the percentage of total ['251]bTSH binding added to each well.
counter.
Results
FTSHr cells - a malignantly transformed rat thyroid cell line
FRTL-5 cells are a well characterized thyroid cell line that is derived from Fisher rat thyroid cells (AmbesiImpiombato et al., 1980) and exhibits many differentiated functions including active iodide transport, thyroglobulin synthesis, TSH-dependent cAMP accumulation
(Vitti et al., 1983) and growth in response to TSH stimulation (recent review: Bidey et al., 1988). These
cells were stably transfected with the human TSH receptor cDNA (Nagayama et al., 1989; Libert et al., 1989). Transfected cells, designated FTSHr cells, acquired a malignant phenotype with a fibroblast-like morphology. In soft agar they displayed an anchorageindependent growth and in vivo they formed malignant tumors when they were transplanted onto nude mice (Derwahl et al., l992a). In contrast to FRTL-5 wild type cells their growth in mice was independent of a TSH enhancing regimen.
In vitro FTSHr cells grew faster than FRTL-5 cells with a doubling time of only 18 h and they had lost the TSH dependency of growth (Derwahi et al., 1992 a). Furthermore, TSH binding to FTSHr cells was observed to be significantly reduced (Figure 1). They still synthesized cAMP in response to TSH, although to a lower extent than FRTL-5 cells, which indicates the presence of a functional TSH receptor-adenylate cyc-
lase system. With respect to differentiated functions Measurement of cAMP accumulation Cells maintained in H5 medium for 5 days were incubated with the indicated concentrations of bovine TSH and 1 mM I methyl-3-isobutylxanthol for 2 h at 37 °C. Then after removal
of the medium cAMP was extracted with 95% ethanol and
intracellular concentrations were measured by a RIA kit (Amersham-Buchler). Results were corrected to the protein content of the cells.
FTSHr cells had lost thyroglobulin synthesis and iodide trapping to TSH stimulation (Figure 2). OÏi the molecular level we demonstrated that FTSHr
cell expressed the rat TSH receptor, albeit at a lower level than FRTL-5 thyroid cells (Derwahl et al., 1992 a). Although several copies of the full length TSH receptor cDNA had been integrated into the genome of FTSHr cells, only a few clones expressed the human
TSH receptor mRNA at a low level. In accordance ['251j iodide uptake FRTL-5 and FTSHr cells were cultured in 24 well plates in H 5 medium supplemented with the indicated TSH concentrations for 3 days. After removal of the medium cells were rinsed twice with PBS and incubated with the same medium supplemented with 0.2 sCl carrier free 125I] (Amersham) at 37 °C for 60 min. Then cells were solubilized with 1 N NaOH and radioactivity was measured in a u-counter. Values were corrected to the protein content of the cells.
with this we found that malignantly transformed cells only bound 20-30% of TSH compared to FRTL-5 thyroid cells (Figure 1).
HTC-TSHr cells - a human thyroid carcinoma cell line
The human thyroid carcinoma cell line HTC was derived from a thyroid follicular carcinoma (Goretzki
Downloaded by: University of Pittsburgh. Copyrighted material.
Transfection of FRTL-5 and HTC cells
M. Broecker et al., TSH and Thyroid Carcinomas
59
HTc-TsHr
OO FRTL5 cells
A-A
4000
HTC
FTSHr 1 cells 3000
2000
1000
0.10
1.0
10
0.01
0.1
1.0
10
100
TSH [mU/mI]
Fig. 1 Specific [1251]bTSH binding to FRTL-5 thyroid cells and malignantly transformed FTSHr cells. Cells were grown in 24 well plates to confluence. Studies of ['251]bTSH binding were performed in presence of the indicated unlabeled TSH concentrations in modified Hank's buffer without NaCI supplemented with 280mM sucrose to maintain isotonicity. The data are expressed as the percentage of total [125IJbTSH binding
added to each well. Mean of 3 independent experiments in duplicate
Ito
100
TSR (mU/mi)
FORSKOUN ruM]
Fig. 3 TSH-dependent cAMP synthesis in HTC wild type and HTC-TSHr cells (on the left). On the right side forskolinstimulated cAMP accumulation in HTC cells is shown indicating a functional adenylate cyclase-system. Mean ± SEM of 3 independent experiments in duplicate.
mRNA and synthesized a functional TSH receptor. As shown in Figure 3 HTC-TSHr cells accumulated cAMP in response to TSH via the re-expressed TSH receptor,
while HTC wild type cells that lack an endogenous receptor did not respond.
Table 1 Inhibitory effect of TSH, dibutyryl-cAMP and forskolin on growth of HTC-TSHr cells. For studies of [3H] thymidine incorpation the cells were cultured in 96 well plates in H 5 medium supplemented with the indicated concentrations FRTL-5 E
6000
Q-
o
of TSH or dibutyryl-cAMP or forskolin for 48 h. Note the weak, but significant inhibitory effect of TSH on growth of HTC-TSHr cells (p < 0.01 for H5 medium supplemented with TSH 100 mU/ml vs. control (H5 medium)). The results are the mean ± SEM of eight measurement
q)
Treatments
D -'Q-
3H] thymidine incorporation (cmp X 100/well)
2000 FTSHr 1
100
10
1000
15H (mU/ L) Fig. 2 Lack of iodide trapping in FTSHr cells. While differentiated FRTL-5 thyroid cells concentrated iodide in response to TSH, malignantly transformed FTSHr cells had lost this ability. Mean ± SEM of 3 independent experiments in duplicate
et al., 1989). In culture these cells lost important differentiated functions such as TSH receptor expression and TSH-stimulated iodide accumulation. We stably transfected the human TSH receptor cDNA into these cells (Derwahl et al., 1992b). The transfected cells (HTC-TSHr cells)
re-expressed
the TSH receptor
Control
257 ± 13.8
TSH 0.1 mU/ml
244 ± 14.9 225 ± 12.1
10 mU/ml 100 mU/ml
db-cAMP 1 tM 10 11M
Forskolin 5 1iM
218 ± 8.3
226± 7.1 142± 8.8 168 ± 9.4
HTC-TSHr cells grew in 5H medium supplemented with 10% fetal calf serum, but also in medium without any hormones and growth factors. Interestingly, TSH displayed a slight, but significant inhibitory effect on proliferation of these cells (Table 1). This inhibition These cells were derived from the differentiated FTC-l33 thyroid carcinoma cell line that had been established by Dr. Goretzki, Düsseldorf, and were kindly provided by him.
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0.01
60
Exp. Clin. Endocrinol. loo (1992) 1/2
was mediated by the cAMP pathways and could be mimicked by forskolin, a stimulator of the adenylate
thyroid carcinomas TSH may exhibit an inhibitory
cyclase, and by dibutyryl-cAMP, a cAMP analog. The same inhibitory effect of both compounds was observed in HTC wild type cells demonstrating that this effect is
tumors.
rather than a stimulatory effect on proliferation of these
a heritable property rather than an acquired trait of
Further studies are necessary to elucidate the role of TSH in controlling growth of differentiated thyroid carcinomas. In a subset of these carcinomas which either
HTC-TSHr cells.
grow TSH-independently or in which growth is inhibited by TSH, the benefit of a TSH-suppressive therapy may have to be reconsidered.
Discussion
Acknowledgments
growth-promoting effect of TSH is dependent on insulin-like growth factor I (IGF I) or insulin as a cofactor
(Roger et al., 1988; Tramontano et al., 1988). However, the role of TSH in growth regulation of thyroid carcinomas is still a controversial topic (Clark, 1981; Cady, 1983). There is strong evidence that most dif-
The expert assistance of Sabine Hoppe and Heike Sippel is gratefully acknowledged. This work was supported by a grant of the Deutsche Forschungsgemeinschaft, Bonn, Germany, to M.D. (De 407/2-1).
References [1] Abe, Y.; Ichikawa, Y.; Homma, M.; Ito, K.; Urata, T.: TSH receptor and adenylate cyclase in undifferentiated thyroid carcinoma. [Letter] Lancet 2 (1977) 506
ferentiated thyroid carcinomas express and synthezise a functional TSH receptor that is coupled to the Gs protein-adenylate cyclase pathway (Saltiel et al., 1981; Brabant et al., 1991; Ohta et al., 1991).
[2] Brabant, G.; Maenhaut, C.; Koehrle, J.; Scheumann, G.; Dralle, H.; Hoang-Vu, C.; von zur Mühlen, A.; Vassart, G.; Dumont, J. E.: Mol. Cell. Endocrinol. 82 (1991) R7-12
In support of these findings we demonstrated that
Meissner, W. A.; Werber, J.; Gelman, R. S.: The effect of thyroid hormone administration upon survival in patients with differentiated thyroid carcinoma. Surgery 94 (1983) 978-983 [4] Clark, O. H.; Castner, B. J.: Thyrotropin receptors in normal and neoplastic human tissue. Surgery 85 (1978)
malignantly
transformed
FTSHr thyroid
cells
do
express the TSH receptor mRNA. In comparison to nontransformed parental FRTL 5
thyroid cells this
expression is decreased as this has been demonstrated in differentiated human carcinomas (Ohta et al., 1991).
Although FTSHr cells synthesize a functional TSH receptor that is coupled to Gs protein-adenylate cyclase system, TSH does not influence growth of these cells. This result calls in question the assumption that either the finding of TSH receptor expression or of specific TSH binding or TSH-stimulated cAMP accumulation in human thyroid carcinomas prove that TSH is a growth factor in these tumors. The same problem was also addressed in our second model, the human thyroid carcinoma cells HTC-TSHr. This cell line was established by transfection of the human TSH receptor into a thyroid carcinoma cell line that had lost TSH receptor expression. Transfection of these cells
resulted in a re-expression of the TSH
receptor and coupling of this receptor to the adenylate cyclase pathway. In contrast to FTSHr cells, growth of HTC-TSHr cells was influenced by TSH. Interestingly, TSH displayed a inhibitory and not a stimulatory effect on growth of these cells. Recently, a similiar observation was reported by Müller-Gärtner and co-workers (1989) who found that in a subset of papillary thyroid
carcinomas TSH displayed an inhibitory effect on growth. Among 15 carcinomas, TSH stimulated proliferation in 4, decreased in 3 and failed to influence in 8 cases. Therefore, it is likely that in vivo in a few
[3] Cady, B.; Cohn, K.; Rossi, R. L.; Sedwick, C. E.;
624-632 [5] Clark, O. H.: TSH suppression in the management of thyroid nodules and thyroid cancer. Wld. J. Surg. 5 (1981) 39-47 [6] Crue Jr., G.: Endocrine dependency of papillary carcinomas of the thyroid. JAMA 195 (1966) 101-110
[7] Crile, G.: Treatment of cancer of thyroid with desiccated thyroid. Cleveland Clin. Q 22 (1955) 161-163
[8] Derwahl, M.; Broecker, M.; Aeschimann, S.; Schatz, H.; Studer, H.: Malignant transformation of rat thyroid cells transfected with the human TSH receptor cDNA. Biochem.
Biophys.
Res.
Commun.
183
(l992a)
220-226 [9] Derwahi, M.; Broecker, M.; Meyer, K.; Schatz, H.: Lower growth rate of a human follicular thyroid carcinoma cell line stably transfected with the human TSH re-
ceptor cDNA. J. Endocrinol. Invest. Suppl. 2 (1992b) 11
1101 Dumont, J. E.: The action of thyrotropin on thyroid metabolism. Vitam. Horm. 29 (1971) 287-295
Feigner, P. L.; Gader, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Danielsen, M.: Proc. Natl. Acad. Sci. USA 84 (1987) 7413-7417 Goretzki, P. E.; Frilling, A.; Simon, D.; Rastegar, M.; Ohmann, C.: Growth regulation of human thyrocytes by thyrotropin, cyclic adenosine monophosphate, epidermal growth factor and insulin-like growth factor. In: Growth
regulation of thyroid gland and thyroid tumors. Eds. Goretzki, P. E.; Röher, H. D.; Vol. 18, Front. Mormon. Res., Basel, Karger Verlag, 1989, pp. 56-80 Libert, F.; Lefort, A.; Gerard, C.; Parmentier, M.; Per-
Downloaded by: University of Pittsburgh. Copyrighted material.
generally accepted that in normal and benign human thyroid tissue TSH is the main regulator of growth and function in vitro and in vivo (Dumont, 1971; Roger et al., 1988; Studer et al., 1989). The It is
M. Broecker et al., TSH and Thyroid Carcinomas
sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding of autoantibodies. Biochem.
ate cyclase and protein phosphokinase activities in human thyroid. Comparison of normal glands, hyperfunctional nodules and carcinomas. Eur. J. Cancer 12 (1976)
Biophys. Res. Commun. 165 (1989): 1250 1255
447-453
Mazzaferri, E. L.: Papillary and follicular thyroid cancer: a selective approach to diagnosis and treatment. Annu. Rev.
Schorr, I.; Minshaw, H. T.; Cooper, M. A.; Mahafee, K.; Ney, R. L.: Adenyl cyclase hormone responses of certain human endocrine tumors. J. Clin. Endocrinol. Metab. 34 (1972) 447-452 Studer, H.; Peter, H. J.; Gerber, H.: Natural heterogeneity
Med. 32 (1981) 73-91 Müller-Gärtner, H. W.; Baisch, H.; Garn, M.; de la Roche,
J.: Individually different proliferation responses of differentiated thyroid carcinomas to thyrotropin. In: Growth
of thyroid cells: the basis for understanding thyroid function
regulation of thyroid gland and thyroid tumors. Eds. Goretzki, P. E.; Röher, H. D.; Vol. 18, Front. Hormon.
and nodular goiter growth. Endocr. Rev. 10 (1989) 125 135
Res., Basel, Karger Verlag, 1989, pp. 137-151 Nagayama, Y.; Kaufman, K. D.; Seto, P.; Rapoport, B.: Molecular cloning, sequence and functional expression of the eDNA for the human thyrotropin receptor. Biochem. Biophys. Res. Commun. 165 (1989) 1184-1190
Takahashi, H.,; Jiang, N. S.; Gorman, C. A.; Lee, C. Y.: Thyrotropin receptors in normal and pathological thyroid tissue. J. Clin. Endocrinol. Metab. 47 (1978) 870-876
Ohta, K.; Endo, T.; Onaya, T.: The mRNA levels of thyrotropin receptor, thyroglobulin and thyroid peroxidase in neoplastic human thyroid tissues. Biochem. Biophys.
Res. Commun. 174 (1991) 1148-1153
Roger, P.; Taton, M.; van Sande, J.; Dumont, J. E.: Mitogenic effects of thyrotropin and adenosine 3',S'-
Tramontano, D.; Ingbar, S. H.: Properties and regulation of the thyrotropin receptor in the FRTL-5 rat thyroid cell line. Endocrinology 118 (1986) 1945-1951 Tramontano, D.; Moses, A. C.; Veneziani, B. M.; Ingbar, S. H.: Adenosine 3' 5 '-monophosphate mediates both the mitogenic effect of thyrotropin and its ability to amplify the response to insulin-like growth factor I in FRTL-5 cells.
Endocrinology 122 (1988) 127 133
monophosphate in differentiated normal thyroid cells in vitro. J. Clin. Endocrinol. Metab. 66(1988)1158-1165
Saltiel, A. R.; Powell-Jones, C. H. J.; Thomas, C. G.; Nayfeh, S. N.: Thyrotropin receptor-adenylate cyclase function in human thyroid neoplasms. Cancer Res. 41 (1981) 2360-2365
Author's address: Priv.-Doz. Dr. M. Derwahl, Medizinische Universitätsklinik, Bergmannsheil, Gilsingstr. 14, W-4630
Sand,. G.; Jortay, A.; Pochaet, R.; Dumont, J. E.: Adenyl-
Bochum, Germany
Downloaded by: University of Pittsburgh. Copyrighted material.
ret, J.; Ludgate, M.; Dumont, J. E.; Vassart, G.: Cloning,
61
Experimental and Clinical Endocrinology © 1992 Johann Ambrosius Barth
Thyrotropin Receptor and Growth of Thyroid Carcinomas M. Broecker' and M. Derwahi
Key words: TSH receptor - Thyroid carcinomas - Growth - Adenylate cyclase
Zusammenfassung: Aufgrund klinischer Erfahrungen wird allgemein angenommen, daß Thyreotropin (TSH) das Wachstum differenzierter Schilddrüsencarcinome stimuliert. Nicht alle klinischen Studien konnten jedoch diese Annahme bestätigen.
Ebenfalls widersprüchlich sind In-vitro- und In-vivo-
Untersuchungen an Transplantaten menschlicher Schilddrüsencarcinome, die entweder einen stimulierenden oder einen hem-
menden oder keinen TSH-Effekt auf das Tumorwachstum nachwiesen. Als gesichert gilt, daß der TSH-Rezeptor in differenzierten Schilddrüsencarcinomen exprimiert wird. Um auf molekularer Ebene die Bedeutung von TSH und seines Rezeptors an Zeilmodellen studieren zu können, etablierten wir zwei permanente Schilddrüsencarcinomzellinien. Durch Transfektion der humanen TSH-Rezeptor-cDNA in FRTL-5-Schilddrüsenzellen entstand die FTSHr-Tumorzellinie, die in vitro und
Thyrotropin (TSH) is widely believed to influence
als Transplantat auf Nacktmäusen TSH-unabhängig wächst, wichtige differenzierte Funktionen (Thyreoglobulinsynthese, Jod-Aufnahme) verloren hat, aber weiterhin ein funktionsfähiges TSH-Rezeptor-Adenylat-Zyklase-System besitzt. Die zweite Tumorzellinie HTC-TSHr wurde durch Transfektion der
humanen TSH-Rezeptor-cDNA in menschliche Schilddrüsencarcinomzellen, die keinen TSH-Rezeptor mehr exprimieren, geschaffen. Diese Zellinie reexprimiert den TSH-Rezeptor, der an das zelleigene Adenylat-Zyklase-System gekoppelt ist. Überraschenderweise führt eine TSH-Stimulation dieser Zellen zu einer Hemmung des Zellwachstums. Beide Tumorzellinien
belegen, daß der alleinige Nachweis einer TSH-RezeptormRNA-Expression, einer spezifischen TSH-Bindung oder einer TSH-abhängigen zyklo-AMP-Synthese nicht ausreichen, eine wachstumsstimulierende TSH-Wirkung abzuleiten. Ferner zeigt sich, daß TSH auch das Wachstum menschlicher Schilddrüsencarcinomzellen hemmen kann.
growth of thyroid carcinomas (Mazzaferri, 1981). This
expressed in differentiated carcinomas, albeit at a lower level than compared to normal thyroid tissue (Brabant et
view is supported by several clinical studies which
al., 1991;Ohtaetal., 1991).
revealed a positive therapeutic effect of a TSH-suppres-
To further elucidate the role of TSH and its receptor in thyroid carcinomas we established two in vitro models, the FTSHr cell line (Derwahl et al., 1992a) and the HTC-TSHr cell line (Derwahl et al., 1992 b).
sive treatment after ablative therapy in patients with thyroid carcinomas (Crile, 1955; Clark, 1981). However, other investigators were unable to confirm these results (Cady, 1983). Various in vitro studies performed during the last two
decades did not resolve this issue. In comparison with normal thyroid tissue TSH binding andlor TSH-stimu-
Materials and Methods
lated cAMP accumulation were found either to be identi-
Cell cultures
cal or increased or decreased in differentiated thyroid carcinomas (Crile, 1966; Schorr et al., 1972; Takahashi et al., 1978; Sand et al., 1976; Abe et al., 1977; Clark et al., 1978; Saltiel et al., 1981). However, it has been demonstrated recently that the TSH receptor mRNA is 1
Part of doctoral thesis at the University of Bochum
Monolayer cultures of cell lines were grown in Coon's modi-
fied Ham F12 medium supplemented with 10% fetal calf serum (HTC cells) or 5% calf serum (FRTL-5 cells) and six hormones or growth factors [glycyl-histidyl-lysine, 10 ng/ml; insulin, 10 sg/mI; somatostatin, 10 ng/ml; transferrin, 5 tg/ml; hydrocortisone, 3.2 ng/mIl with 10 mU/ml (H6-medium) or without bovine TSH (H 5-medium), 100 UI penicillinlml and 100 sg streptomycin/mI in 25 cm2 plastic culture flasks (Corn-
Downloaded by: University of Pittsburgh. Copyrighted material.
Laboratories of Endocrinology, University Clinic of Internal Medicine Bergmannsheil, Bochum, Germany
Exp. Clin. Endocrinol. loo (1992) 1/2
58
ing; New York, NY). Cells were passaged by enzymatic dissociation with trypsin EDTA (Seromed, Biochrom, Berlin,
[3H] thymidine incorporation
Germany).
For studies of [3H] thymidine incorporation the cells were cultured in 96 well plates in H 5 medium supplemented with the indicated concentrations of TSH or dibutyryl-cAMP or forskolin for 48 h. Then cells were removed from plates by treatment with trypsin and measured in a j3 scintillation
The human TSH receptor cDNA (hTSHR-cDNA) inserted into the Eco RI sites of the eukaryotic expression plasmid pSV2-NEO-ECE (kindly provided by Dr. B. Rapoport, San Francisco) was transfected into FRTL-5 cells and HTC cells using a modified lipofection method (Felgner et al., 1987). Briefly, plasmid DNA was mixed with cationic liposomes (10 sl per .tg DNA) and the formed DNA/liposome complex applied to a 100 mm culture dish. The monolayer cultures were incubated for 4 hours and then regular culture medium was added. 4-5 days after transfection 400 sg/ml Geneticin (0418) was added to the culture medium to select 1
transfected cells. After 4-5 weeks when non-transfected cells had died, cells were passaged and surviving clones were pooled. Transformed cells of three independent transfections were further subcloned by limiting dilution. As negative control, cells were transfected with the plasmid pSV 2-NEO-ECE without the hTSHR-cDNA.
Radiolabeled TSH binding
FTSHr cells were grown in 24 well plates to confluence. Binding studies were performed in Hank's modified buffer without NaCl (Tramontano et al., 1986). To maintain isotonicity 280 mM sucrose was added. Highly purified '25I]bTSH was a generous gift of Dr. Herzog (Henning Ber-
lin, Germany). Cells were incubated for 2 h at 37 °C with ['25IIbTSH and the indicated amount of unlabeled TSH (Sigma). At the end of the incubation cells were rinsed three times with the same buffer (4C) and solubilized with I N NaOH. Radioactivity was measured in a u-counter. Nonspecific '25I}bTSH was determined in presence of 10_6 M unlabeled TSH, and this value subtracted from total binding. The data are expressed as the percentage of total ['251]bTSH binding added to each well.
counter.
Results
FTSHr cells - a malignantly transformed rat thyroid cell line
FRTL-5 cells are a well characterized thyroid cell line that is derived from Fisher rat thyroid cells (AmbesiImpiombato et al., 1980) and exhibits many differentiated functions including active iodide transport, thyroglobulin synthesis, TSH-dependent cAMP accumulation
(Vitti et al., 1983) and growth in response to TSH stimulation (recent review: Bidey et al., 1988). These
cells were stably transfected with the human TSH receptor cDNA (Nagayama et al., 1989; Libert et al., 1989). Transfected cells, designated FTSHr cells, acquired a malignant phenotype with a fibroblast-like morphology. In soft agar they displayed an anchorageindependent growth and in vivo they formed malignant tumors when they were transplanted onto nude mice (Derwahl et al., l992a). In contrast to FRTL-5 wild type cells their growth in mice was independent of a TSH enhancing regimen.
In vitro FTSHr cells grew faster than FRTL-5 cells with a doubling time of only 18 h and they had lost the TSH dependency of growth (Derwahi et al., 1992 a). Furthermore, TSH binding to FTSHr cells was observed to be significantly reduced (Figure 1). They still synthesized cAMP in response to TSH, although to a lower extent than FRTL-5 cells, which indicates the presence of a functional TSH receptor-adenylate cyc-
lase system. With respect to differentiated functions Measurement of cAMP accumulation Cells maintained in H5 medium for 5 days were incubated with the indicated concentrations of bovine TSH and 1 mM I methyl-3-isobutylxanthol for 2 h at 37 °C. Then after removal
of the medium cAMP was extracted with 95% ethanol and
intracellular concentrations were measured by a RIA kit (Amersham-Buchler). Results were corrected to the protein content of the cells.
FTSHr cells had lost thyroglobulin synthesis and iodide trapping to TSH stimulation (Figure 2). OÏi the molecular level we demonstrated that FTSHr
cell expressed the rat TSH receptor, albeit at a lower level than FRTL-5 thyroid cells (Derwahl et al., 1992 a). Although several copies of the full length TSH receptor cDNA had been integrated into the genome of FTSHr cells, only a few clones expressed the human
TSH receptor mRNA at a low level. In accordance ['251j iodide uptake FRTL-5 and FTSHr cells were cultured in 24 well plates in H 5 medium supplemented with the indicated TSH concentrations for 3 days. After removal of the medium cells were rinsed twice with PBS and incubated with the same medium supplemented with 0.2 sCl carrier free 125I] (Amersham) at 37 °C for 60 min. Then cells were solubilized with 1 N NaOH and radioactivity was measured in a u-counter. Values were corrected to the protein content of the cells.
with this we found that malignantly transformed cells only bound 20-30% of TSH compared to FRTL-5 thyroid cells (Figure 1).
HTC-TSHr cells - a human thyroid carcinoma cell line
The human thyroid carcinoma cell line HTC was derived from a thyroid follicular carcinoma (Goretzki
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Transfection of FRTL-5 and HTC cells
M. Broecker et al., TSH and Thyroid Carcinomas
59
HTc-TsHr
OO FRTL5 cells
A-A
4000
HTC
FTSHr 1 cells 3000
2000
1000
0.10
1.0
10
0.01
0.1
1.0
10
100
TSH [mU/mI]
Fig. 1 Specific [1251]bTSH binding to FRTL-5 thyroid cells and malignantly transformed FTSHr cells. Cells were grown in 24 well plates to confluence. Studies of ['251]bTSH binding were performed in presence of the indicated unlabeled TSH concentrations in modified Hank's buffer without NaCI supplemented with 280mM sucrose to maintain isotonicity. The data are expressed as the percentage of total [125IJbTSH binding
added to each well. Mean of 3 independent experiments in duplicate
Ito
100
TSR (mU/mi)
FORSKOUN ruM]
Fig. 3 TSH-dependent cAMP synthesis in HTC wild type and HTC-TSHr cells (on the left). On the right side forskolinstimulated cAMP accumulation in HTC cells is shown indicating a functional adenylate cyclase-system. Mean ± SEM of 3 independent experiments in duplicate.
mRNA and synthesized a functional TSH receptor. As shown in Figure 3 HTC-TSHr cells accumulated cAMP in response to TSH via the re-expressed TSH receptor,
while HTC wild type cells that lack an endogenous receptor did not respond.
Table 1 Inhibitory effect of TSH, dibutyryl-cAMP and forskolin on growth of HTC-TSHr cells. For studies of [3H] thymidine incorpation the cells were cultured in 96 well plates in H 5 medium supplemented with the indicated concentrations FRTL-5 E
6000
Q-
o
of TSH or dibutyryl-cAMP or forskolin for 48 h. Note the weak, but significant inhibitory effect of TSH on growth of HTC-TSHr cells (p < 0.01 for H5 medium supplemented with TSH 100 mU/ml vs. control (H5 medium)). The results are the mean ± SEM of eight measurement
q)
Treatments
D -'Q-
3H] thymidine incorporation (cmp X 100/well)
2000 FTSHr 1
100
10
1000
15H (mU/ L) Fig. 2 Lack of iodide trapping in FTSHr cells. While differentiated FRTL-5 thyroid cells concentrated iodide in response to TSH, malignantly transformed FTSHr cells had lost this ability. Mean ± SEM of 3 independent experiments in duplicate
et al., 1989). In culture these cells lost important differentiated functions such as TSH receptor expression and TSH-stimulated iodide accumulation. We stably transfected the human TSH receptor cDNA into these cells (Derwahl et al., 1992b). The transfected cells (HTC-TSHr cells)
re-expressed
the TSH receptor
Control
257 ± 13.8
TSH 0.1 mU/ml
244 ± 14.9 225 ± 12.1
10 mU/ml 100 mU/ml
db-cAMP 1 tM 10 11M
Forskolin 5 1iM
218 ± 8.3
226± 7.1 142± 8.8 168 ± 9.4
HTC-TSHr cells grew in 5H medium supplemented with 10% fetal calf serum, but also in medium without any hormones and growth factors. Interestingly, TSH displayed a slight, but significant inhibitory effect on proliferation of these cells (Table 1). This inhibition These cells were derived from the differentiated FTC-l33 thyroid carcinoma cell line that had been established by Dr. Goretzki, Düsseldorf, and were kindly provided by him.
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0.01
60
Exp. Clin. Endocrinol. loo (1992) 1/2
was mediated by the cAMP pathways and could be mimicked by forskolin, a stimulator of the adenylate
thyroid carcinomas TSH may exhibit an inhibitory
cyclase, and by dibutyryl-cAMP, a cAMP analog. The same inhibitory effect of both compounds was observed in HTC wild type cells demonstrating that this effect is
tumors.
rather than a stimulatory effect on proliferation of these
a heritable property rather than an acquired trait of
Further studies are necessary to elucidate the role of TSH in controlling growth of differentiated thyroid carcinomas. In a subset of these carcinomas which either
HTC-TSHr cells.
grow TSH-independently or in which growth is inhibited by TSH, the benefit of a TSH-suppressive therapy may have to be reconsidered.
Discussion
Acknowledgments
growth-promoting effect of TSH is dependent on insulin-like growth factor I (IGF I) or insulin as a cofactor
(Roger et al., 1988; Tramontano et al., 1988). However, the role of TSH in growth regulation of thyroid carcinomas is still a controversial topic (Clark, 1981; Cady, 1983). There is strong evidence that most dif-
The expert assistance of Sabine Hoppe and Heike Sippel is gratefully acknowledged. This work was supported by a grant of the Deutsche Forschungsgemeinschaft, Bonn, Germany, to M.D. (De 407/2-1).
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ferentiated thyroid carcinomas express and synthezise a functional TSH receptor that is coupled to the Gs protein-adenylate cyclase pathway (Saltiel et al., 1981; Brabant et al., 1991; Ohta et al., 1991).
[2] Brabant, G.; Maenhaut, C.; Koehrle, J.; Scheumann, G.; Dralle, H.; Hoang-Vu, C.; von zur Mühlen, A.; Vassart, G.; Dumont, J. E.: Mol. Cell. Endocrinol. 82 (1991) R7-12
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malignantly
transformed
FTSHr thyroid
cells
do
express the TSH receptor mRNA. In comparison to nontransformed parental FRTL 5
thyroid cells this
expression is decreased as this has been demonstrated in differentiated human carcinomas (Ohta et al., 1991).
Although FTSHr cells synthesize a functional TSH receptor that is coupled to Gs protein-adenylate cyclase system, TSH does not influence growth of these cells. This result calls in question the assumption that either the finding of TSH receptor expression or of specific TSH binding or TSH-stimulated cAMP accumulation in human thyroid carcinomas prove that TSH is a growth factor in these tumors. The same problem was also addressed in our second model, the human thyroid carcinoma cells HTC-TSHr. This cell line was established by transfection of the human TSH receptor into a thyroid carcinoma cell line that had lost TSH receptor expression. Transfection of these cells
resulted in a re-expression of the TSH
receptor and coupling of this receptor to the adenylate cyclase pathway. In contrast to FTSHr cells, growth of HTC-TSHr cells was influenced by TSH. Interestingly, TSH displayed a inhibitory and not a stimulatory effect on growth of these cells. Recently, a similiar observation was reported by Müller-Gärtner and co-workers (1989) who found that in a subset of papillary thyroid
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Author's address: Priv.-Doz. Dr. M. Derwahl, Medizinische Universitätsklinik, Bergmannsheil, Gilsingstr. 14, W-4630
Sand,. G.; Jortay, A.; Pochaet, R.; Dumont, J. E.: Adenyl-
Bochum, Germany
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ret, J.; Ludgate, M.; Dumont, J. E.; Vassart, G.: Cloning,
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