Effects of Thyroid Hormone on Androgen

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[CANCER RESEARCH 51. 4323-4327, August 15. 1991] Effects of Thyroid Hormone on Androgen- or Basic Fibroblast Growth Factor-induced Proliferation of Shionogi Carcinoma 115 Mouse Mammary Carcinoma Cells in Serum-free Culture1

Satoru Sumitani, Soji Kasayama, Takahisa MirÃ³se,Keishi Matsumoto, and Bunzo Sato2

The Third Department of Internal Medicine [S. S., S. K., T. H., B. S.J and The Second Department of Pathology [K. M.Â¡,Osaka University Hospital, Fukushima-ku 1-1-50, Osaka 553, Japan

ABSTRACT (GC cells) has also been observed to be enhanced by thyroid hormone (9, 10). Thyroid hormone receptor belongs to the DNA synthesis of SC-3 cells cloned from mouse mammary carcinoma steroid/thyroid/vitamin receptor superfamily (11). Thyroid (Shionogi carcinoma 115) was remarkably enhanced by androgen as well as basic fibroblast growth factor (bFGF) at the early phase (days 1-3) hormone might possibly modulate steroid hormone actions. of stimulation in serum-free culture condition. However, bFGF-induced Actually, interactions between thyroid and steroid hormones DNA synthesis could not be observed at the late phase (days 4-6) of have been reported with respect to the control of neoplastic stimulation while androgen was able to continuously elicit DNA synthesis. growth (12). In this relation, thyroid hormone has been reported When the effect of androgen on cell yield was examined, the cell number to inhibit the growth of Shionogi carcinoma cell line in FCS- was increased while bFGF could not enhance cell growth. Androgen- supplemented culture (13, 14). PCS has been known to contain induced heparin-binding growth factor partially purified from conditioned various growth control factors which may complicate the inter medium behaved like bFGF in terms of DNA synthesis and replication pretation of the data on growth-stimulatory or -inhibitory ef in SC-3 cells. fects of thyroid hormone. SC-3 cells were found to contain the high-affinity binding site toward The present experiments were designed to study the effects triiodothyronine. The dissociation constant and the maximum number of the binding sites were 7 x 10~'Â°Mand 1800/cell, respectively. Triiodo of androgen, bFGF and/or thyroid hormone on SC-3 cell thyronine significantly blunted the testosterone-induced DNA synthesis. growth in serum-free culture. The results show that androgen On the other hand, bFGF-enhanced DNA synthesis was not substantially stimulates both DNA synthesis and proliferation of SC-3 cells inhibited by triiodothyronine. while bFGF enhances only the DNA synthesis without increas These results suggest that androgen, but not bFGF, has unique action ing the cell yield. Thyroid hormone suppresses effectively the site(s) which might be important for SC-3 cell replication and might be growth-stimulatory ability of androgen but less effectively that antagonized by thyroid hormone. of bFGF.

INTRODUCTION MATERIALS AND METHODS Materials. MEM and Ham's F-12 medium were obtained from Shionogi carcinoma 115, which is a spontaneously developed mouse mammary carcinoma, is one of the most well-character Nissui Pharmaceutical Co., Ltd. (Tokyo. Japan). FCS was from Hy- ized androgen-dependent tumors. The growth of SC-1153 is Clone Laboratories, Inc. (Logan, UT). Testosterone, bovine serum albumin (essentially fatty acid free), Norit A, T3, thyroxine, monoio- markedly enhanced by androgen in vivo (1). The clonal cell line termed as SC-3, derived from SC-115, is also growth stimulated dotyrosine, and diiodotyrosine were from Sigma Chemical Co. (St. Louis, MO). Bovine brain-derived bFGF was purchased from R & D by androgen in serum-free culture (2). This androgen-induced Systems, Inc. (Minneapolis, MN). Heparin-Sepharose was from Phar growth has recently been demonstrated to be mediated through macia (Piscataway, NJ). Ampure SA and Ampure DT were obtained an induction of HBGF (3). The growth-stimulatory ability of from Amersham Japan (Tokyo, Japan), [methyl-sH]Thym\dine (specific androgen-induced HBGF can be blocked by anti-bFGF anti activity, 70-90 Ci/mmol), [3'-'"I]T3 (specific activity, 315 Ci/mmol), body (4). Furthermore, our recent experiment would suggest and D-threo-[dichloroacetyl-l,2-'4C]chloramphenicol (60 mCi/mmol) that this androgen-induced HBGF is able to be associated with (CAT assay grade) were obtained from New England Nuclear (Cam FGF receptor (5). In line with these observations, only FGFs bridge, MA). The restriction enzymes were from Toyobo Co., Ltd. among growth factors examined have been found to enhance (Tokyo, Japan). The other reagents used here were of analytical grade. Cell Culture. SC-3 cells were cloned from SC-115 (2) and were DNA synthesis of androgen-unstimulated SC-3 cells (6). How maintained in MEM supplemented with 2% FCS and IO"8 M ever, it remains to be elucidated whether or not the biological testosterone. potency of androgen is qualitatively or quantitatively different Cell Growth Experiment. The cell growth experiment was conducted from that of bFGF. as reported previously (6) with a minor modification. SC-3 cells from Thyroid hormone has been known to exert profound effects a stock culture were plated on 24-well plates (10" cells/well) containing on gene activation (7, 8). The growth of cultured pituitary cells 1 ml MEM supplemented with 2% DCC-treated FCS (no testosterone). DCC treatment was found to effectively remove T3 from FCS to less Received 1/22/91; accepted 6/4/91. than 10~" M (data not shown). On the following day (day 0), SC-3 cells The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in were washed with HMB medium (serum-free medium) and cultured in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. HMB medium containing various test compounds. These media were 1This investigation was supported by grants-in-aid from the Ministry of changed every' other day. The cell number was measured on the indi Education, Tokyo, Japan, the Cancer Research Promotion Fund, Tokyo, Japan, cated day by a Coulter Counter. the Foundation of the Growth Science and the Hirai Cancer Research Fund. 2To whom requests for reprints should be addressed. DNA Synthesis. SC-3 cells were plated on 96-well plates (2,000 or 'The abbreviations used are: SC-115, Shionogi carcinoma 115; bFGF, basic 10,000 cells/well) containing 0.15 ml MEM supplemented with 2% fibroblast growth factor; HBGF, heparin-binding growth factor: T3, triiodothy DCC-treated FCS. On the following day, SC-3 cells were washed with ronine; PCS, fetal calf serum; DCC. dextran-coated charcoal: MEM, Eagle's minimum essential medium; HMB medium. Ham's F-12: MEM (1:1, v/v) con and cultured in HMB medium without any stimulant for 24 h to taining 0.1% (w/v) bovine serum albumin: TRE, thyroid hormone responsive minimize S-phase cells in cultures. Then, SC-3 cells were stimulated element; tk, thymidine kinase; CAT, chloramphenicol acetyl transferase. with various test compounds for the indicated periods of time. To 4323

DNA (20 fig). The cuvettes were exposed twice to the electroporation procedure (400-420 V; 25 Â¿iF).Eachexposure was followed by cooling at 0Â°Cfor 10 min. These cells were then plated onto 60-mm culture dishes in 5 ml MEM supplemented with 10% DCC-treated FCS in the presence or the absence of T3. After 48-h cultures, the cell extracts were subjected to CAT assay as described previously (17). Statistical Analysis. The data presented here were expressed as mean Â±SE. A paired Student's t test was used to discuss the significant difference.

RESULTS 246 Difference in Growth-stimulatory Effects on SC-3 Cells be Days in Culture tween Testosterone and bFGF. When testosterone was added in Fig. 1. Effects of testosterone, bFGF, or HBGF on replication of SC-3 cells. HMB medium at a concentration of 10~8M, the number of SC- SC-3 cells were plated in MEM supplemented with 2% DCC-treated FCS and stimulated with 10~" M testosterone (A), 1 ng/ml bFGF (â€¢),or HBGF (Ãœ)for 3 cells was increased as described previously (6, 15) (Fig. 1). In the indicated periods of time. The unstimulated samples (O) were also included. contrast, bFGF failed to stimulate cell growth. Androgen-in The cell number of triplicate samples was measured. duced HBGF also failed to stimulate SC-3 cell replication. Next, their growth-stimulatory ability to initiate DNA synthesis measure the DNA synthesis, the cells were pulsed with ['HJthymidine was measured. Both testosterone and bFGF markedly enhanced (0.3 ^Ci/well) at 37Â°Cfor 2 h. Radioactivity incorporated into the cells the incorporation of [3H]thymidine into the cells at the early was measured as described previously (15). phase of stimulation (days 1-3) (Fig. 2). At the later phase Preparation of Androgen-induced Heparin-binding Growth Factor. SI (days 4-6), bFGF-induced DNA synthesis almost disappeared. 3 cells were plated on 100-mm dishes (IO6 cells/dish) containing 10 ml This phase-dependent alteration in bFGF ability could not be MEM supplemented with 2% DCC-treated FCS and 10~8M testoster modified even when a high concentration (10 ng/ml) was used one. On the following day (day 0), the medium was changed to HMB to stimulate SC-3 cells. Partially purified HBGF secreted from medium supplemented with 10~8M testosterone. On day 1, the medium testosterone-stimulated SC-3 cells again stimulated DNA syn was changed with fresh HMB medium containing IO"8 M testosterone thesis in a manner analogous to that of bFGF. and conditioned for 24 h. The conditioned medium (approximately Effects of Tj on Testosterone- or bFGF-induced SC-3 Cell 1000 ml) was filtered and applied to a column of heparin-Sepharose which had been preequilibrated with 10 mM Tris-HCl (pH 7.0 at 20Â°C)- Growth. The ability of T3 to modulate SC-3 cell growth was then examined (Fig. 3). The cells were cultured in the presence 0.1 % (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1 -propanesul- of 10~8 M testosterone with various concentrations of T3, and fonate buffer (5). After being washed with the equilibration buffer, materials bound to the column were eluted with 20 ml 2 M NaCl in the the cell number was measured on day 6. T3 inhibited the equilibration buffer. 3-[(3-Cholamidopropyl)-dimethylammonio]-l- proliferation of testosterone-stimulated SC-3 cells in a dose- propanesulfonate in the eluate was removed by Ampure DT column. dependent manner. The cell yield was inhibited maximally at Subsequently the eluate was desalted by Ampure SA column to HMB 10~8 M T3 (Fig. 3). Next, T3 effects on testosterone- or bFGF- medium and processed for the estimate of growth-promoting activity. induced enhancement of DNA synthesis were examined on day Analyses of Nuclear T3 Receptor. SC-3 cells were cultured in MEM containing 2% DCC-treated FCS and 10~8M testosterone and then in 1. The representative results were shown in Fig. 4A. T3 inhibited HMB medium supplemented with IO"8 M testosterone for 24 h up to DNA synthesis of testosterone-stimulated SC-3 cells. In con subconfluence. After being washed with HMB medium, SC-3 cells were trast, bFGF-induced DNA synthesis was not substantially influ incubated with various concentrations of [I25I]T3,usually for 90 min at enced by T3. Four separate experiments revealed that T3-de- 37Â°Cin 95% air/5% CO2 (16), which was found to be sufficient to pendent inhibition of DNA synthesis in testosterone-stimulated achieve an equilibrium of T3 in SC-3 cells (see below). To assess the cells is statistically significant (P < 0.01), but not in bFGF- nonspecific binding, a 100-fold excess of radioinert T3 was added to stimulated cells. The cells prestimulated with testosterone or parallel samples. To examine the ligand specificity, SC-3 cells were bFGF for 6 days were also examined (Fig. 4B). DNA synthesis incubated with 0.2 nM [125I]T3inthe presence of various concentrations of competitors as described above. After incubations, SC-3 cells were harvested in 5 ml solubilization buffer (20 mM Tris-HCl, pH 7.85, at 20Â°C,1 mM MgCl2, 0.5% Triton X-100) with a rubber policeman and transferred to plastic tubes. After standing for 30 min at 4Â°C,asucrose concentration was adjusted to 0.25 M, and the tubes were centrifuged at 1500 x g for 15 min at 4Â°C.Nuclear pellets were washed 3 times with 1 ml buffer (0.25 M sucrose-20 miMTris-HCl, pH 7.85 at 20Â°C). The radioactivity associated with washed nuclei was measured by a Packard gamma counter. These binding data obtained were normalized with the cell number used for incubations with [125I]T3. Construction of TRE-tk-CAT Reporter Plasmici and Transfection. The reporter plasm id (TRE-tk-CAT gene) was constructed essentially as described before ( 17). Briefly, the CAT sequence and tk promoter were inserted to pUC 18 after the corresponding plasmids were digested with appropriate restriction enzymes. Synthetic oligonucleotide which con Fig. 2. Effects of testosterone, bFGF, or HBGF on the DNA synthesis of SC- tains two tandem of TRE (18) was ligated to the BamHl site upstream 3 cells. SC-3 cells (2 x I03/well) were plated in MEM supplemented with 2% of pUC 18-tk-CAT (Fig. 8). After being washed with (0.272 M sucrose- DCC-treated FCS and then cultured in HMB medium with or without various 7 mM phosphate-1 mM MgCI2, pH 7.4 at 20'C) buffer, SC-3 cells (2.7 stimulants for the indicated periods of time. To examine the effect of autocrine growth factor, the partially purified and concentrated (20-fold) HBGF was mixed x IO7cells/ml) were transferred into Gene Pulser Cuvettes (Bio-Rad, with HMB medium at a final concentration of 2%. SC-3 cells were then pulse- Richmond, CA) containing the reporter plasmid (20 Mg)and calf thymus labeled with [3H]thymidine. Symbols as in Fig. 1. 4324

rosine as well as diiodotyrosine could not exert their inhibitory 100 ability up to 10~6 M. Thus, the nuclear binding site for T3 can be classified as T3 receptor. The ability of T3 analogues to inhibit DNA synthesis of testosterone-stimulated SC-3 cells was also addressed (Fig. 7). Thyroxine did inhibit the DNA synthesis less effectively than T3. Diiodotyrosine showed inhi bition to a lesser extent, while monoiodotyrosine failed to exhibit the inhibitory action. To confirm the biological ability of T3 receptor in SC-3 cells, the cells were transfected with the TRE-tk-CATgene and stimulated with T3. As illustrated in Fig. 8, CAT activity was clearly enhanced in response to T3 stimuli, indicating that T3 receptor in SC-3 cells is able to activate T3- responsive genes. 10 10 Concentrations of T3 (M) DISCUSSION Fig. 3. Inhibition of testosterone-induced SC-3 cell replication by T3. SC-3 cells (1 x 104/well) stimulated with IO"8 M testosterone were cultured in the The present report describes the differential responsiveness presence of various concentrations of T3 for 6 days, and the cell number of of SC-3 cells to testosterone and bFGF in serum-free culture triplicate samples was determined.

PH ] Thymidine Incorporation {dpm/well X10'-*)

â€¢o 8- o io" io" io* io* io' 10* [3H ] Thymidine Incorporation (dpm/well X10'4) Concenti-moni Ã¶lT3[M]

-â€¢â€”â€¢â€¢--bFGF I 61

Fig. 4. Effects of T3 on DNA synthesis of testosterone- or bFGF-stimulated SC-3 cells. SC-3 cells (2 x 103/well) stimulated with 1(T8 M testosterone or 10 ng/ml bFGF were cultured in the presence of IO"8 M T3 for 24 h (A) or 6 days X 4- -o- T (B). Unstimulated SC-3 cells (C) or cells stimulated with 10~8 M T3 alone (7"3) were also examined. These cells were pulse-labeled with [3H]thymidine. Repre sentative results were obtained by triplicate assay. Additional separate experi ments (three for A and two for B) gave similar results (see text). id11 id10 io" io8 io7 16* of testosterone-stimulated cells was again significantly (three Concentrations of T3 [M] separate experiments; P < 0.01) inhibited by T3. Dose-response Fig. 5. Dose-response study of T3-dependent inhibition of testosterone- or studies on T3-dependent inhibition of DNA synthesis were also bFGF-induced DNA synthesis of SC-3 cells. SC-3 cells (1 x 104/well) were plated addressed on day 1 (Fig. 5). The dose-dependent inhibition by and cultured in the presence of various concentrations of T3 with 10~8M testos terone or 10 ng/ml bFGF for 24 h. Their ability to synthesize DNA was measured Tj was again observed in testosterone-stimulated cells, while as described in "Materials and Methods." SE values were less than 5% at all DNA synthesis elicited by a high concentration (10 ng/ml) of points. Inst't. percentage of the maximum, taking the values obtained in the bFGF was only marginally inhibited by T3. absence of T3 as 100%. Analyses of T3 Receptor in SC-3 Cells. Since T3 receptor is an obligatory component for T3 to exert its action, the nuclear binding site for T3 in SC-3 cells was analyzed. When the cells were incubated for the various periods of time, the amount of [125I]T3specifically associated with nuclei reached maximum levels at 30 min, which were maintained for at least 120 min (Fig. 6A). In the following experiments, therefore, the cells were incubated for 90 min to analyze the T3 receptor. Scatchard analyses revealed the presence of a high-affinity T3 binding site (Fig. 6Ã„).The dissociation constant and the maximum number of the binding site were 6.7 Â±1.6 x 10~'Â°Mand 1780 Â±310 sites/cell (n = 3), respectively. The ligand binding specificity CononlraUoru of T3â€¢ruloguM(M) was also examined. As shown in Fig. 6C, the radioinert T3 Fig. 6. Analyses of the T3 receptor in SC-3 cells. |125I]T3binding assays were could effectively inhibit specific [125I]T3binding to nuclei while performed as described in "Materials and Methods." The kinetic data on [12!I]T3 specifically associated with nuclei (A), the Scatchard plot (B), and the ligand thyroxine less effectively inhibited this binding. Monoiodoty- specificity (C). 4325

in response to a removal of testosterone without any cell syn chronization pressure. Thus, the serum-free culture system enabled us to clarify the real potencies of testosterone or bFGF in the process of SC-3 cell growth. Â»_MIT Thyroid hormones have been known to positively (9, 10, 22) or negatively (13,14, 23) regulate cell growth. GC cells, a clonal cell line derived from a rat pituitary tumor, have been reported to be growth stimulated by T_, in the culture containing the resin-treated serum (9, 10). This T3-induced enhancement of the replication rate was proved to be due to the decrease in the duration of the G, phase of the cell cycle. The growth of MCF- 7 cells (human breast cancer cell line) was also stimulated by T3 in serum-free but growth-modulating factor-supplemented culture condition (22). In contrast, cell lines from SC-115 have been found to show a growth-inhibitory response to T3 (Refs. 13 and 14 and this study). This T3-dependent growth inhibition in SC-115 cells does not seem to be an exceptional case since Concentrations of T3 analogues (M) the replication of SFME cells, a mouse embryo cell line, is also Fig. 7. Effects of Tj analogues on the DNA synthesis of testosterone-stimulated inhibited by T, (23). These growth-stimulatory and -inhibitory SC-3 cells. SC-3 cells (1 x I04/Â»ell)were plated and cultured in HMB medium containing 10~*M testosterone plus various concentrations of T, analogues for 3 abilities of T3 are not surprising since estrogen also exhibits days and pulse-labeled with | Ã•1jiln inuline Each value was obtained by triplicate both positive and negative effects on tumor growth (24). Partic assay. ularly, thyroid hormones have been known to exert diverse actions as permissive hormones (25). For example, thyroid condition. Testosterone stimulates both DNA synthesis and hormones have been reported to regulate mRNA levels for the G-protein ÃŸ-subunit,which is involved in the signal transduc- replication, whereas bFGF enhances only DNA synthesis at the early phase of stimulation without the enhanced replication tion of many stimulants (26). In addition, the molecular mech rate. The disappearance of the ability of bFGF or androgen- anisms of thyroid hormone-dependent inhibition of gene induced HBGF to stimulate DNA synthesis at the late phase expression have been recently proposed (27, 28). Since SC-3 cells contain functional Tj receptor, it can be of cultures might not be due to their instability, since the high concentration (10 ng/ml) of bFGF could not elicit a continuous speculated that thyroid hormone action on cell growth may be response in terms of DNA synthesis. Although further studies mediated through nuclear T3 receptor. This notion would be are required to completely purify HBGF, sodium dodecyl sul- supported by the finding that the ability of Tj analogues to fate-polyacrylamide gel electrophoresis of our preparations re suppress testosterone-induced DNA synthesis is in parallel with vealed that only a couple of bands were identified on a silver- their ability to be associated with T3 receptor. However, the stained gel (data not shown; also see Ref. 5). relatively high concentrations of T3 are required to exert its Combined with our previous results (3), the present obser vation would indicate that testosterone exerts an action other than an induction of HBGF in terms of SC-3 cell growth. The molecular mechanism remains to be elucidated for testosterone to commit the cells to the replication state. To our knowledge, however, this is the first report that sex hormone-dependent replication processes are regulated by at least two different mechanisms. These observations are also compatible with a general concept that bFGF and its related molecules are clas sified as a so-called competence factor (19). A competence factor alone is known to be unable to elicit DNA synthesis. Therefore, bFGF- or HBGF-induced DNA synthesis observed at the early phase of cultures might occur in concert with the residual effects of FCS which were used to maintain and plate SC-3 cells. In this relation, bFGF has been observed to mark edly enhance the replication of SC-3 cells in a medium supple mented with 1% DCC-treated FCS (20). In addition, our pre vious results revealed that bFGF exhibits the cell replication potency on SC-3 cells when the cells are plated at the high cell density (4). These observations would suggest that SC-3 cells may secrete not only HBGF but also additional growth-modu lating factor(s) whose synthesis is enhanced by testosterone.

Among hormonal steroids, estrogen has been most extensively (THIi)Â»-lk CAT studied in relation to its growth-stimulatory ability (for a review, see Ref. 21). However, the mechanism of estrogen-induced Fig. 8. Tj receptor-mediated expression of the TRE-lk-CAT gene transfected into SC-3 cells. SC-3 cells were transfected with the TRE-lk-CAT gene followed shortening of the cell cycle is far from conclusive. This may be by culturing with ( 7",)or without (C) 10~8M T, for 48 h. The CAT activity of the due to the fact that many culture systems use FCS. An addi cellular extracts was measured as described in "Materials and Methods." Bottom, tional advantage to our system is that the cells become quiescent structure of the TRE-tk-CA T gene. 4326

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1991 American Association for Cancer Research. THYROID HORMONE AND ANDROGEN SENSITIVE CANCER growth-inhibitory action in SC-3 cells, being similar to the 8. Zilz. N. D., Murray. M. B.. and Towle. H. C. Identification of multiple thyroid hormone response elements located far upstream from the rat Su observation by Natoli et al. (13) and Sica et al. (14). Recently, promoter. J. Biol. Chem., 265: 8136-8143, 1990. a spectrum of biological responses has been reported to be 9. DeFesi, C. R., Fels, E. C., and Surks. M. I. Triiodothyronine stimulates growth of cultured GC cells by action early in the G, period. Endocrinology, regulated by distinct T3 dose ranges, indicating that the impact 114: 293-295, 1984. of Tj nuclear receptor occupancy on T3 responses might be 10. Gingrich, S. A., Smith, P. J., Shapiro, L. E., and Surks, M. I. 5,5'-Diphe- m Ululanti mi (phenytoin) attenuates the action of 3.5,3'-triiodo-l-thyronine variable (29). A similar observation has been published in glucocorticoid regulation of c-Jun function and mouse mam in cultured GC cells. Endocrinology, 116: 2306-2313. 1985. 11. Evans. R. M. The steroid and thyroid hormone receptor superfamily. Science mary tumor virus promotor activation, both of which are the (Washington DC), 240: 889-895. 1988. direct targets of glucocorticoid receptor complexes (30). Thus, 12. Sorrentino. J. M.. Kirkland. W. L., andSirbask. D. A. Control of cell growth. II. Requirement of thyroid hormones for the in vivo dependent growth of rat we would like to believe that T3 action on SC-3 cell growth is pituitary tumor cells. J. Nati. Cancer Inst., 56: 1155-1158, 1976. mediated through its interaction with the T3 receptor. 13. Natoli, C, Sica, G.. Natoli, V., Scambia, G., and Jacobelli. S. Growth The action site(s) of T3 in regulating the growth of SC-3 cells inhibitory effects of thyroid hormones on androgen-dependent mammary tumor cells. J. Steroid Biochem.. 15: 409-413. 1981. is worthwhile discussing. T3 stimulation did not result in a 14. Sica, G., Natoli, C., Marchetti, P.. and Jacobelli, S. Effect of thyroid decrease in the number of androgen receptors in SC-3 cells hormones on androgen responsiveness in a mammary tumor cell line. J. (data not shown). Although the degree of T3-dependent inhibi Steroid Biochem., IS: 415-419. 1981. 15. Hiraoka, D., Nakamura, N., Nishizawa, Y., Uchida, N., Noguchi, S., Mat tion of SC-3 cell growth varied among the experiments, testos sumoto, K.. and Sato. B. Inhibitory and stimulatory effects of glucocorticoid terone-induced growth was more profoundly inhibited by T3 on androgen-induccd grow th of murine Shionogi carcinoma 115 in rim and in cell culture. Cancer Res., 47: 6560-6564, 1987. than bFGF-induced growth. Also, testosterone-induced repli 16. Burke. R. E., and McGuire, W. L. Nuclear thyroid hormone receptors in a cation was suppressed by T3. These results might suggest that human breast cancer cell line. Cancer Res.. 38: 3769-3773, 1978. T3 exerts its action at the site where testosterone commits SC- 17. Miyashita. Y., Hirose. T.. Kouhara. H.. Kishimoto, S., Matsumoto. K.. and Sato. B. Identification of unoccupied but transformed nuclear estrogen 3 cells to the replication state. Thus, T3 can be used as a receptor in cultured mouse Leydig cell. J. Steroid Biochem.. 35: 561-567, valuable tool to differentiate the action sites of testosterone in 1990. SC-3 cell growth. In this relation, T3 has been reported to exert 18. Beato. M. Gene regulation by steroid hormones. Cell, 56: 335-344, 1989. 19. Stiles, C. D.. Capone. G. T., Scher, C. D., Antoniades, H. N., Van Wyk, J. its growth-stimulatory effects on GC cells early in the GI period J., and Pledger, W. J. Dual control of cell growth by somatomedins and (9, 10). This stimulation of GC cell growth by T3 required the platelet-derived growth factor. Proc. Nati. Acad. Sci. USA, 76: 1279-1283. 1979. synthesis of proteins, some of which were identified in the 20. Kasayama, S.. Sumitani. S.. Tanaka. A.. Yamanishi, H., Nakamura, N., culture medium as autocrine or paracrine growth factors (31). Matsumoto. K.. and Sato. B. Heparin inhibits autocrine stimulation but not The molecular nature of T,-induced growth factor secreted from FGF stimulation of cell proliferation of androgen-responsive Shionogi car cinoma 115. J. Cell. Physiol.. in press, 1991. GC cells is not well characterized. The serum-free culture is 21. Lippman. M. E., Dickson. R. B.. Bates. S.. Knabbe. C., Huff, K., Swain, S., well suited for characterization of the biochemical effects of T3 McManaway, M.. Bronzert. D.. and Kasid, A. Autocrine and paracrine on T3-sensitive cells. These interesting but unresolved problems growth regulation of human breast cancer. Breast Cancer Res. Treat., 7: 59- 70, 1986. in SC-3 cells should be clarified by further studies. 22. Barnes, D., and Sato, G. Growth of a human mammary cancer cell line in serum free medium. Nature (Lond.). 2Ã„/:3823-3829, 1978. 23. Loo. D.. Rawson, C., Schmitt, M., Lindburg. K., and Barnes, D. Glucocor ticoid and thyroid hormones inhibit proliferation of serum-free mouse em REFERENCES bryo (SFME) cells. J. Cell. Physiol.. 142: 210-217, 1990. 24. Sato, B., Maeda. Y.. Nishizawa, Y., Noma. K.. Kishimoto. S.. and Matsu 1. Minesita, T., and Yamaguchi, K. An androgen-dependem mouse mammary moto. K. Effects of tamoxifen on estrogen-induced enhancement or inhibition tumor. Cancer Res., .25 1168-1175. 1965. of tumor growth in mouse Leydig cell tumor lines. Cancer Res.. 44: 3286- 2. Noguchi, S., Nishizawa. Y., Nakamura, N., Uchida. N.. Yamaguchi. K.. Sato, B., Kitamura, Y., and Matsumoto, K. Growth-stimulating effect of pharma 4391, 1984. 25. Malbon. C. C.. Rapiejko, P. J., and Watkins. D. C. Permissive hormone cological doses of estrogen on androgen-dependent Shionogi carcinoma 115 regulation of hormone-sensitive effector systems. Trends Pharmacol. Sci., 9: in vivo but not in cell culture. Cancer Res., 47: 263-268, 1987. 33-36. 1988. 3. Sato, B., Nakamura, N., Noguchi. S., Uchida. N.. and Matsumoto. K. 26. Rapiejko. P. J., Watkins, D. C, Ros, M., and Malbon, C. C. Thyroid Characterization of androgen-dependent autocrine growth factor secreted hormones regulate G-protein /i-subunit mRNA expression in viro. J. Biol. from mouse mammary carcinoma (Shionogi carcinoma 115). in: H. Imura, Chem., 26* 16183-16189. 1989. K. Shizume, and S. Yoshida (eds.). Progress in Endocrinology 1988, pp. 99- 27. Chatterjee, V. K. K.. Lee, J-K., Rentoumis. O.. and Jameson, J. L. Negative 104. Amsterdam, The Netherlands: Elsevier Science Publishers B.V.. 1988. regulation of the thyroid stimulating hormone gene by thyroid hormone: 4. Lu, }.. Nishizawa, Y, Tanaka, A., Nonomura, N.. Yamanishi, H., Uchida, receptor interaction adjacent to TATA box. Proc. Nati. Acad. Sci. USA, 86: N., Sato, B., and Matsumoto, K. Inhibitory effect of antibody against basic 9114-9118.1989. fibroblast growth factor on androgen- or glucocorticoid-induced growth of 28. Holloway. J. M.. Glass, C. K., Adler, S., Nelson, C. A., and Rosenfeld. M. Shionogi carcinoma 115 cells in serum-free culture. Culture Res.. 49:4963- G. The C-terminal interaction domain of the thyroid hormone receptor 4967, 1989. confers the ability of the DNA site to dictate positive or negative transcrip- 5. Nonomura, N., Lu, J., Tanaka, A., Yamanishi. H.. Sato, B., Sonoda. T., and tional activity. Proc. Nati. Acad. Sci. USA, 87: 8160-8164. 1990. Matsumoto, K. Interaction of androgen-induced autocrine heparin-binding 29. Halperin, Y., Surks, M. I., and Shapiro. L. E. L-Triiodothyronine (T3) growth factor with fibroblast growth factor receptor on androgen-dependent regulates cellular growth rate, growth hormone production, and levels of Shionogi carcinoma 115 cells. Cancer Res., 50: 2316-2321. 1990. nuclear Tj receptors via distinct dose-response ranges in cultured GC cells. 6. Nakamura, N., Yamanishi. H., Lu, J., Uchida, N., Nonomura, N.. Matsu Endocrinology. 726: 2321-2326. 1990. moto, K., and Sato, B. Growth-stimulatory effects of androgen, high concen 30. Jonat. C., Rahmsdorf, H. J.. Park, K-K., Cato, A. C. B., Gebel. S., Ponta, tration of glucocorticoid or fibroblast growth factors on a cloned cell line H., and Herrlich, P. Antitumor promotion and antiinflammation: down- from Shionogi carcinoma 115 cells in a serum-free medium. J. Steroid modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell, 62: Biochem., 33: 13-18, 1989. 1189-1204,1990. 7. Petty, K. J., Desvergne. B., Mitsuhashi. T.. and Nikodem. V. M. Identifica 31. Miller, M. J.. Fels. E. C., Shapiro, L. E., and Surks, M. I. L-Triiodolhyronine tion of a thyroid hormone response element in the malic enzyme gene. J. stimulates growth by induction of an autocrine/paracrine growth factor. J. Biol. Chem., 265: 7395-7400, 1990. Clin. Invest.. 79: 1773-1781, 1987.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1991 American Association for Cancer Research. Effects of Thyroid Hormone on Androgen- or Basic Fibroblast Growth Factor-induced Proliferation of Shionogi Carcinoma 115 Mouse Mammary Carcinoma Cells in Serum-free Culture

Satoru Sumitani, Soji Kasayama, Takahisa Hirose, et al.

Cancer Res 1991;51:4323-4327.

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