Molecular and Ce&dar Endocrinology, 44 (1986) 195.199

Elsevier Scientific Publishers Ireland, Ltd. 195

MCE 01430

Control of growth in cultured rat thyroid cells

Willard H. Dere and Basil Rapoport Departments of Medicine, L4. Medical Center. ~etabolj~rn (II IF). 4150 Clement Street, San Fran~~~c~ CA 94f2f, and the Vnioers~t~ of

California, San Fran&co, CA (V. So A.)

(Received 10 June 1985; accepted 8 November 1985)

Key words: TSH; thyroid cells; cyclic AMP.

Summary

The purpose of this study was to determine the role of CAMP in the growth of FRTL and FRTL, cells, 2 continuous cultured thyroid lines. TSH, at concentrations similar to those reported to induce growth in primary dog thyroid cultures, played an essential role for growth. Stimulators of adenylate cyclase, cholera toxin and forskolin, and CAMP analogues, dibuty~l CAMP and Et-bromo CAMP, mimicked the effect of TSH in both groups of cultured cells. The present data confirm the role of TSH in controlling growth of both cell lines and suggest that CAMP is an essential intracellular mediator of TSH action.

Introduction

There is conflicting evidence regarding the role of cyclic AMP in the control of mammalian cell replication in general. Several reviews correlate high intracellular CAMP levels with growth arrest, thus supporting the notion that CAMP plays an inhibitory role in growth (Boynton and Whitfield, 1983; Ralph, 1983). Rozengurt et al. on the other hand have demonstrated the growth-promoting ef- fect of CAMP on mouse 3T3 cells (Rozengurt, 1981; Rozengurt et al., 1981). In addition, other investigators have provided evidence that transi- tory cAMP elevations during key Gl and G2 phases of the cell cycle are essential for cellular growth (Boynton and Whitfield, 1983).

A similar controversy exists regarding the role of CAMP in thyroid cell replication. Roger and Dumont have suggested a positive role for CAMP in the growth of dog thyroid cells in primary culture by providing evidence that the mitogenic effect of thyrotropin (TSH) is CAMP-dependent (Dumont et al., 1981; Roger and Dumont, 1984). Valente et al., by contrast, have reported that

TSH-induced FRTL, thyroid cell division and CAMP production are independent functions in that cell growth was not mimicked by dibutyryl CAMP, a CAMP analogue (Valente et al., 1983a). Whether or not CAMP induces thyroid cell divi- sion is also central to the thesis that thyroid- stimulating and thyroid-growth immunoglobulins exist as separate entities in autoimmune thyroid disease (Doniach et al., 1982; Valente et al., 1983b). This is because if CAMP does indeed mediate thyroid growth, it is difficult to conclude that these 2 antibodies are distinctly different. The present studies were conducted to reexamine this important issue, and demonstrate that CAMP does play an import~t role in thyroid cellular division.

Materials and methods

The materials used and the companies from which they were purchased are listed: thyrotropin (Armour Pharmaceutical, Phoenix, AZ); dibutyryl cyclic AMP, 8-bromo cyclic AMP, sodium buty- rate, 3-isobutyl-l~methylx~thine, calf thymus

0303-7207/86/$03.50 0 1986 EIsevier Scientific Publishers Ireland, Ltd.

196

DNA, human transferrin, Hoechst No. 33258 (his-Benzimide) (Sigma Chemical Company, St. Louis, MO); forskolin (CalBiochem-Behring, La Jolla, CA); trypsin, Dulbecco’s phosphate-buffered saline, calf serum (University of California, Cell Culture Facility, San Francisco, CA); F-12 media (K.C. Biological, Lenexa, KS); insulin (Squibb Novo, Princeton, NJ).

Culture of rat thyroid ceil Fischer rat thyroid (FRTL) cells are a line of

rat thyroid cells in continuous culture that retain the principal differentiated characteristics of thy- roid follicular cells (Ambesi-Impiombato et al., 1980). FRTL, cells are a variant line derived from FRTL cells. These cells maintain the same dif- ferentiated features as FRTL cells but have a more rapid rate of division. FRTL, have been adapted to grow at a higher calf serum concentration (5%). Both cell lines are totally dependent on TSH for growth (Ambesi-Impiombato et al., 1980). Cells were grown in Coon’s modified Ham’s F-12 medium supplemented with insulin (10 pg/ml), transferrin (5 pg/ml), TSH (1 mu/ml) (3H medium), and ~tibiotics-penicillin (125 U/ml), gentamicin (40 pg/ml), and amphotericin B (2.5 r_lg/ml). Medium was replaced every 3-4 days. For indi~dual experiments, one dish (100 mm diameter) of cells was subcultured into 3.5 mm diameter cluster dishes (Falcon No. 3046). For subculture, the cells were washed twice with Dulbecco’s phosphate-buffered saline (PBS), calci- um- and magnesium-free. Three ml of 0.05% tryp- sin in PBS with 0.02% EDTA were added and the dishes were incubated at 37°C for 5-10 min. The resuspended cells were diluted in 3H medium, and plated at low density in 35 mm diameter cluster dishes (approximately 2-5 x lo4 cells/well). The cells were cultured for 48 h at 37°C in a water- saturated, water-jacketed incubator in 5% CO, in air in preparation for the experiments described in the text.

Following the 48 h incubation period, during which time the FRTL or FRTL, cells adhered to the bottom of the dishes, the cells were washed 2 times with Dulbecco’s PBS with calcium and mag- nesium. Cells in duplicate or triplicate wells were maintained in TSH-free medium (2H medium) supplemented with different agents, as indicated in

the text for individual experiments. Medip were replaced at 2-3-day intervals. Cells were harvested at 3-5-day intervals over a 2-week period for determination of their rate of growth. Each well was washed 3 times with Dulbecco’s PBS after which 0.75 ml of 1 N NaOH was added. Wells were scraped with a rubber policeman and the solubilized cells were transferred to 12 x 75 mm tubes and kept at -20°C until assayed for their DNA content.

DNA assay The sensitive DNA assay of Labarca and Paigen

(1980) was used to determine DNA concentrations in the FRTL and FRTL, specimens. The principle of the assay is as follows: a fluorochrome, Hoechst 33258, reacts with, and binds to, single-str~ded DNA causing fluorescence which is linear over a range of DNA concentrations. Hoechst 33258 was diluted to a concentration of 0.5 pg/ml in 2 M NaCl, 0.2 M NaH,PO,, pH 7.0. Solubilized cell samples were heated at 60°C for 30 min to dissoci- ate double-stranded DNA, and then immediately chilled to 4’C to prevent re-annealing of the DNA strands. NC1 (1 N) was added to the reaction to neutralize the NaOH, and Hoechst 33258 was added. The tubes were vortexed and fluorescence measured in a Perkins-Elmer fluorometer (excita- tion 356 nm, emission 458 nm). Sample DNA concentrations were determined against standards of calf thymus DNA (0, 1, 2, 3, 4 and 5 pg/tube).

Results

Preliminary experiments were performed to de- termine the dose-response relations~p between TSH and FRTL cell growth. In the absence of TSH no cellular replication occurred (Fig. 1). Stimulaton of FRTL replication was initially evi- dent at a TSH con~ntration of 0.05 mu/ml and was near maximal at only 0.10 mu/ml {Fig. l), indicating a remarkably steep dose-response rela- tionship.

In order to test whether CAMP mimics the action of TSH in stimulating FRTL cell repli- cation, cells were exposed for up to 2 weeks to CAMP analogues or agents that are known to enhance cAMP generation in intact cells. For- skolin (10 PM), cholera toxin (0.1 pg/ml), di-

TSH

L-I W/ml) 0.55

/

0.50

0.10

1 0.05

0 CONTROL 5 10 15

DAYS

Fig. 1. TSH effect on FRTL cell growth. FRTL cells were incubated for 15 days in 2H media alone (control) or with TSH at the concentrations indicated. Each point represents the meanf SD of thyroid cellular DNA content obtained in trip- licate culture dishes.

tm CUOLERA TOXIN

A-----A CUOLERA TOXIN

I&X 0 DIRUTYRVL CAMP

I&X

CONTROL

2 4 6 8’ 10 12 14

DAYS

3

32 7

9

9 0 1

I-

t

197

i------o FORSKOLtN

DAYS

Fig. 2. Forskolin and FRTL cell growth. FRTL cells were incubated for 14 days in 2H media containing TSH (0.1 mu/ml) or forskolin (10 PM), with or without 3-isobutyl-l-methyl- xanthine (0.5 mM). Control cells were grown in 2H media alone. Each point represents the mean f SD of thyroid cellular DNA content obtained in triplicate culture dishes.

I T

5-

1 4- .I

5 3-

2 2-

l-

o- IJ CON- m

Fig. 4. CAMP and FRTL, cell growth. FRTL, cells were incubated in 2H media alone (control) or 2H media containing the indicated additives. Each bar represents the mean f SD of thyroid cellular DNA content obtained in triplicate culture dishes after 12 days of growth.

Fig. 3. Cholera toxin and dibutyryl CAMP and FRTL cell growth. FRTL cells were incubated for 14 days in 2H media containing TSH (0.1 mu/ml), cholera toxin (0.1 pg/mf), or dibutyryl CAMP (1 mM), with or without 3-isobutyl-l-methyl- xanthine (0.5 mM). Control cells were grown in 2H media alone. Each point represents the mean f SD of thyroid cellular DNA content obtained in triplicate culture dishes.

198

butyryl CAMP (1 mM) (Figs. 2 and 3) and 8bromo CAMP (0.3 mM) (data not shown) all stimulated FRTL cell growth. Furthermore, the extent of cellular growth was generally comparable to that seen in TSH-stimulated cells. Su~~singly, how- ever, 3-isobutyl-l-methylxanthine, a phospho- diesterase inhibitor that inhibits CAMP degrada- tion, greatly attenuated the FRTL growth response to agents that raised intracellular CAMP content (Figs. 2 and 3).

The foregoing studies were conducted with FRTL cells because FRTL, cells were not initially available. At a later stage when we obtained FRTL, cells it was important to confirm our prior ob- servations with these cehs. Similar data were ob- tained with both cell types. Thus, after 12 days of incubation, TSH (1 mU/mI), forskolin (100 PM), cholera toxin (0.1 ,ug/ml), and dibutyryl CAMP (1 mM) all stimulated cell growth (Fig. 4). TSH 0.1 mu/ml caused approximately 60% as much growth as the higher TSH dose. Forskolin 10 FM tripled cell growth over control, yet increased DNA synthesis only one third as much as did forskolin 100 PM. As a control sodium butyrate did not stimulate FRTL, cell growth (data not shown).

Discussion

Studies with primary dog thyroid cultures and a clonal line of rat thyroid cells (FRTL,) have dem- onstrated the importance of TSH in inducing thyroid cell growth and differentiation (Ambesi- Impiombato et al., 1980; Roger and Dumont, 1984). The puzzling lack of effort (or even inhibi- tion) of TSH on human and porcine thyroid cell growth (Westermark et al., 1979; Risdall et al., 1984) may be explained by the presence of an inhibitor in crude TSH preparations (Valente et al., 1983a,b) or may be related to species dif- ferences. Clearly, therefore, the picture is complex and our data can only be related to rat (and probably dog) thyroid cells. Insulin and TSH work synergistically to trigger DNA synthesis and cellu- lar proliferation in dog thyroid cells (Roger et al., 1983). Controversy exists however as to wether or not TSH-induced growth (when it indeed occurs in rat and dog thyroid cells) is mediated by CAMP. Thus, Roger and Dumont observed that cholera toxin, forskohn, and dibutyryl CAMP (together

with a phosphodiesterase inhibitor) mimicked the effect of TSH (Roger and Dumont, 1984). By contrast, Valente et al. (1983a) reported a dissocia- tion between CAMP content and growth in FRTL, rat thyroid cells. Most interesting and provocative in the latter study was the finding of Valente et al. that there appeared to be 3 subpopulations of patients with Graves’ disease; those whose IgG stimulated both CAMP content and growth in FRTL, cells, those whose IgG induced a high cAMP response with little cellular growth, and those whose IgG stimulated growth with a minimal or absent CAMP response (Valente et al., 1983b).

The present study was designed to re-examine the role of TSH and CAMP in FRTL thyroid ceI1 growth. As in previous studies (Ambesi-Impiom- bato et al., 1980; Valente et al., 1983a), we ob- served an essential role for TSH in the growth of these cells. Thus a minimum concentration of 0.05 mu/ml TSH was necessary to maintain FRTL cells in culture over a 2-week period. The growth response to TSH was very steep. TSH at 0.1 mU/mI induced a near-maximal growth response, with little further stimulation at 0.5 mu/ml. In FRTL, cells, TSH 0.1 mu/ml caused a half-maxi- mal growth response, and 1.0 mu/ml TSH a maxima1 response. These TSH concentrations are similar to those reported by Roger and Dumont with dog thyroid cell cultures (half-maximal and maximal stimulatory concentrations of 0.1 and 1 mU/ml TSH, respectively). Two analogues of CAMP, dibutyryl CAMP and 8-bromo CAMP proved to be potent mitogens of FRTL cells and mimicked the action of TSH. The fact that sodium butyrate did not promote cell growth suggests that CAMP is the active agent in dibutyryl CAMP.

The reason why our data contrast with those of Valente et al., who used the same cell type, is uncertain. However, these investigators did not use the full range of agents that may be employed to examine this question. Thus, forskolin, a diterpene that directly activates adenylate cyclase (Seamon et al., 1981; Darfler et al., 1982), mimics the effect of TSH on thyroid hormone secretion, thyroid cell growth and differentiation in primary dog cultures (Van Sande et al., 1983; Roger and Dumont, 1984). Cholera toxin stimulates thyroid cell adenylate cyclase in a TSH-independent manner by ADP-ribosylation of the stimulatory regulatory

199

subunit N, (Ross et al., 1979). Our data demon- strate that both forskolin and cholera toxin mimic

TSH-induced FRTL and FRTL, cell growth, con- firming reports of similar studies in dog cultures.

It was surprising that 3-isobutyl-l-methyl- xanthine, an inhibitor of intracellular CAMP breakdown, consistently attenuated the growth re- sponse of FRTL cells to all stimulators tested. The reason for this diminished response is not clear.

Methylxanthines are thought to exert at least 2 major effects: (1) inhibition of cyclic nucleotide phosphodiesterases; and (2) competitive inhibition of adenosine binding sites in the adenylate cyclase system (Londos and Wolff, 1977; Londos et al., 1980). Regarding the first possibility, the induc-

tion of cell growth by CAMP may be biphasic, with inhibition of growth occurring at very high CAMP levels. Consistent with this possibility is the previous observation that TSH stimulation of

thyroid cells in the absence of IBMX has a minimal

effect on absolute intracellular CAMP concentra- tions (Dumont et al., 1981) even though the turnover rate of the CAMP pool is presumably greatly increased. In contrast, intracellular CAMP levels rise greatly in response to TSH stimulation when degradation is inhibited by IBMX.

Methylxanthines antagozine primarily the aden- osine R binding site, hence IBMX could cause unopposed P site inhibition of adenylate cyclase

activity. However, its explanation seems very un- likely because the inhibitory effect of IBMX oc- curred in the presence of high concentrations of CAMP analogues. Furthermore, both beef and dog thyroid cells appear to lack an R site (Cochaux et

al., 1982). Finally, our data indicating that CAMP does

indeed mimic TSH action in FRTL, cells raises questions about the nature of the putative thyroid growth immunoglobulin (TGI) in Graves’ disease, in particular whether this is a separate entity from thyroid-stimulating immunoglobulin (TSI). Fun- damental to the thesis that TGI is distinct from TSI is that CAMP does not mediate thyroid cellu- lar growth. This is because 90-95% of IgGs from patients with untreated Graves’ disease have TSI bioactivity (Rapoport et al., 1984). If CAMP

mediates cellular growth it would therefore not be

possible to detect TGI in these samples. TGI could only be clearly differentiated from TSI in IgG

devoid of TSI activity, a small minority of sam-

ples. Further studies are necessary to prove that TGI is a unique and separate antibody.

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