FRTL-5 Cell Proliferation Research Articles

Effects of the rotary cell culture system-simulated microgravity on growth features of thyroid follicular epithelial cells in rats

The rotary cell culture system(RCCS)was used to simulate the microgravity environment, and FRTL-5 cells were divided into simulated microgravity group(SMG)and normal gravity group(NG). FRTL-5 cells were harvested after treatment for 6, 12, 24, and 36 h, the cell viability was measured by MTT assay, and the cells cycles were detected by flow cytometry. The ultrastructure of FRTL-5 cells was observed under laser confocal microscope with FITC-labeled technique. The MTT assay showed that the proliferation of FRTL-5 cells was significantly inhibited after RCCS treatment for 6, 12, 24, and 36h compared with NG(P<0.05), in which the most obvious effect was observed at 24h. The flow cytometry showed that the proportion of FRTL-5 cells at G1 stage in RCCS group was increased significantly after 6, 12, 24, and 36h compared with NG(P<0.05), while the proportion of FRTL-5 cells at S stage was decreased significantly(P<0.05)except that cultured with RCCS for 6 h. The proportion of FRTL-5 cells at G2/M stage was decreased in early phase(6-12 hours)of RCCS culture, with the lowest at 12h and transient increase at 24h of RCCS culture. The laser confocal microscope revealed that there were local microfilament depolymerization, tension fibers decrease, structure disorder, cellular pseudopodia reduction, and irregular shape among FITC-labeled FRTL-5 cells cultured with RCCS for 36h. (Chin J Endocrinol Metab, 2018, 34: 598-601) Key words: Simulated microgravity; Thyroid follicular epithelial cells; FRTL-5 cell line; Proliferation; Microfilament

Significance of the transient receptor potential canonical 2 (TRPC2) channel in the regulation of rat thyroid FRTL-5 cell proliferation, migration, adhesion and invasion

Mammalian transient receptor potential (TRP) channels are involved in many physiologically important processes. Here, we have studied the significance of the TRPC2 channel in the regulation of rat thyroid FRTL-5 cell proliferation, migration, adhesion and invasion, using stable TRPC2 (shTRPC2) knock-down cells. In the shTRPC2 cells, proliferation was decreased due to a prolonged G1/S cell cycle phase. The tumor suppressor p53 and the cyclin-dependant kinase inhibitors p27 and p21 were upregulated. Cell invasion, adhesion and migration were also attenuated in shTRPC2 cells, probably due to decreased activity of both Rac and calpain, and a decreased secretion and activity of matrix metalloproteinase 2. The attenuated proliferation, migration, invasion and ATP-evoked calcium entry was mimicked by overexpressing a non-conducting, truncated TRPC2 (TRPC2-DN) in wild type cells, and was reversed by overexpression of TRPC2-GFP in shTRPC2 cells. In conclusion, TRPC2 is an important regulator of rat thyroid cell function.

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This study investigates the in vitro effect of eleven thyroid-active compounds known to affect pituitary and/or thyroid weights in vivo, using the proliferation of GH3 rat pituitary cells in the so-called "T-screen," and of FRTL-5 rat thyroid cells in a newly developed test denoted "TSH-screen" to gain insight into the relative value of these in vitro proliferation tests for an integrated testing strategy (ITS) for thyroid activity. Pituitary cell proliferation in the T-screen was stimulated by three out of eleven tested compounds, namely thyrotropin releasing hormone (TRH), triiodothyronine (T3) and thyroxine (T4). Of these three compounds, only T4 causes an increase in relative pituitary weight, and thus T4 was the only compound for which the effect in the in vitro assay correlated with a reported in vivo effect. As to the newly developed TSH-screen, two compounds had an effect, namely, thyroid-stimulating hormone (TSH) induced and T4 antagonized FRTL-5 cell proliferation. These effects correlated with in vivo changes induced by these compounds on thyroid weight. Altogether, the results indicate that most of the selected compounds affect pituitary and thyroid weights by modes of action different from a direct thyroid hormone receptor (THR) or TSH receptor (TSHR)-mediated effect, and point to the need for additional in vitro tests for an ITS. Additional analysis of the T-screen revealed a positive correlation between the THR-mediated effects of the tested compounds in vitro and their effects on relative heart weight in vivo, suggesting that the T-screen may directly predict this THR-mediated in vivo adverse effect.

The effects of amitrole on thyroglobulin and iodide uptake in FRTL-5 cells

Thyroid is a frequent target for endocrine effects of pesticides. Thyroglobulin (TG) and iodide uptake are crucial to thyroid hormone synthesis and may be targets of thyroid-disrupting chemicals. In our study, thyroid follicular FRTL-5 cells were treated with amitrole, an inhibitor of the thyroid peroxidase (TPO), and the effects on TG and total iodide uptake were observed. The results showed that 1-100 mg/L amitrole had no marked effects on FRTL-5 cell proliferation and DNA synthesis. However, it significantly increased the transcription of tg gene and inhibited the total iodide uptake. And 10-100 mg/L amitrole significantly decreased TG in the culture medium. The data suggests amitrole may disrupt the expression and secretion of TG and iodide uptake.

Ghrelin enhances the proliferating effect of thyroid stimulating hormone in FRTL-5 thyroid cells

Ghrelin regulates cell proliferation through the growth hormone secretagogue receptor (GHS-R). We confirmed the expression of GHS-R in FRTL-5 thyroid cells and investigated the effects of ghrelin in thyrocytes using FRTL-5 cells. Ghrelin increased intracellular calcium levels but not intracellular cyclic AMP levels. Ghrelin activated Erk within 2min, then activated Akt and STAT3. Erk phosphorylation was inhibited by the calcium inhibitor cyclopiazonic acid (CPA). Ghrelin alone did not stimulate FRTL-5 cell proliferation but enhanced the effects of thyroid stimulating hormone (TSH). Pretreatment with TSH potentiates the growth effects of ghrelin in thyroid cells, and p66Shc, a growth factor receptor adaptor protein, might mediate these synergistic effects. Ghrelin phosphorylated TSH-induced p66Shc, which was inhibited by CPA. Ghrelin did not affect the proliferation of ARO cells, which showed no increased expression of p66Shc after TSH treatment. Thus, ghrelin-induced intracellular calcium signaling enhanced the TSH-induced proliferation of thyrocytes, possibly mediated by the p66Shc pathway.

An Alternative Therapy for Graves' Disease Clinical Effects and Mechanisms of an Herbal Remedy

Graves' disease, the most common cause of hyperthyroidism, is an autoimmune disorder. Antithyroid drugs have been selected as the first-line treatment of Graves' disease in Korea, Japan, and European countries. However, antithyroid drugs such as methimazole (MMI) and prophylthiouracil (PTU) have limitations in clinical applications because of their side effects. In this study, we performed a clinical trial and in vitro study to investigate the clinical effects and action mechanism of Ahnjeonbaekho-tang (AJBHT), an herbal remedy for Graves' disease. In a clinical study of Graves' disease patients who had side effects from antithyroid drugs, we found that treatment by AJBHT resulted in a reduction of serum triiodothyronine (T3) and free thyroxine (FT4) levels and an increase in thyroid stimulating hormone (TSH) levels (T3: p<0.0001, FT4: p=0.0012, TSH: p=0.0370, respectively). In vitro, AJBHT significantly inhibits FRTL-5 cell proliferation, DNA synthesis, cyclic AMP production, T4 synthesis, and the expression of thyroglobulin (Tg) mRNA in comparison with the control. These results suggest that AJBHT might suppress T(4) synthesis by modulating adenosine 3',5'-cyclic monophosphate (cAMP) and Tg expression, and therefore, AJBHT could be an alternative therapy for Graves' disease patients who have side effects from antithyroid drugs.

P38 mitogen-activated protein kinase contributes to cell cycle regulation by cAMP in FRTL-5 thyroid cells

Thyrotropin activates the cAMP pathway in thyroid cells, and stimulates cell cycle progression in cooperation with insulin or insulin-like growth factor-I. Because p38 mitogen-activated protein kinases (p38 MAPKs) were stimulated by cAMP in the FRTL-5 rat thyroid cell line, we investigated (i) the effect of the specific inhibition of p38 MAPKs on FRTL-5 cell proliferation and (ii) the mechanism of action of p38 MAPKs on cell cycle control, by studying the expression and/or the activity of several cell cycle regulatory proteins in FRTL-5 cells. DNA synthesis was monitored by incorporation of [(3)H]thymidine into DNA and the cell cycle distribution was assessed by fluorescence-activated cell sorter analysis. Expression of cell cycle regulatory proteins was determined by Western blot analysis. Cyclin-dependent kinase 2 (Cdk2) activity associated to cyclin E was immunoprecipitated and was measured by an in vitro kinase assay. SB203580, an inhibitor of alpha and beta isoforms of p38 MAPKs, but not its inactive analog SB202474, inhibited DNA synthesis and the G1-S transition induced by forskolin plus insulin. SB203580 inhibited specifically p38 MAPK activity but not other kinase activities such as Akt and p70-S6 kinase. Treatment of FRTL-5 cells with SB203580 decreased total and cyclin E-associated Cdk2 kinase activity stimulated with forskolin and insulin. However, inhibition of p38 MAPKs by SB203580 was without effect on total cyclin E and Cdk2 levels. The decrease in Cdk2 kinase activity caused by SB203580 treatment was not due to an increased expression of p21(Cip1) or p27(Kip1) inhibitory proteins. In addition, SB203580 affected neither Cdc25A phosphatase expression nor Cdk2 Tyr-15 phosphorylation. Inhibition of p38 MAPKs decreased Cdk2-cyclin E activation by regulating the subcellular localization of Cdk2 and its phosphorylation on Thr-160. These results indicate that p38 MAPK activity is involved in the regulation of cell cycle progression in FRTL-5 thyroid cells, at least in part by increasing nuclear Cdk2 activity.

Regulation of geranylgeranyl pyrophosphate synthase in the proliferation of rat FRTL-5 cells: involvement of both cAMP-PKA and PI3-AKT pathways

We have reported that geranylgeranyl pyrophosphate (GGPP), one of the isoprenoids in the mevalonate pathway, plays an essential role for cell growth through the geranylgeranylation of Rho small GTPases, which control the degradation of P27Kip1 at G1/S transition in rat thyroid FRTL-5 cells. Since GGPP is synthesized from isopentenyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP) by GGPP synthase, we analyzed the regulatory roles of GGPP synthase in the proliferation of FRTL-5 cells stimulated by thyrotropin and insulin in the presence of 5% calf serum (TSH + Ins). We found that: (1) GGPP synthase was activated at G1/S transition with increasing mRNA accumulation followed by protein expression, (2) pravastatin, an inhibitor of HMG-CoA reductase, did not suppress the increasing activity of GGPP synthase with its protein expression although it inhibits proliferation in growth-stimulated FRTL-5 cells, (3) forskolin stimulated proliferation with activation of GGPP synthase in FRTL-5 cells, and (4) LY294002, an inhibitor of phosphatidylinositol 3-kinase, inhibited proliferation with the decreasing activity of GGPP synthase in growth-stimulated FRTL-5 cells. These data indicated that growth stimulation by TSH + Ins increased the activity of GGPP synthase with its increasing protein expression from G1/S transition, in which both cAMP-PKA and PI3-kinase pathways are involved in the proliferation of FRTL-5 cells.

Somatostatin Interferes with Thyrotropin-induced G1-S Transition Mediated by cAMP-dependent Protein Kinase and Phosphatidylinositol 3-Kinase: INVOLVEMENT OF RhoA AND CYCLIN E·CYCLIN-DEPENDENT KINASE 2 COMPLEXES

cAMP-mediated cell proliferation is a complex process that involves multiple pathways. Using a cAMP-dependent cell system, FRTL-5 thyroid cells, we have previously demonstrated the existence of a precise autocrine loop in the control of cell proliferation that involves the positive effector thyrotropin (TSH) and the general inhibitor somatostatin. In search of the regulatory mechanisms responsible for the TSH and somatostatin control of cell proliferation, we analyzed the cell cycle regulatory proteins and the cellular pathways involved in the action of both signals. The results show that specific inhibition of cAMP-dependent protein kinase (PKA) and phosphatidylinositol (PI) 3-kinase blocks independently TSH-induced FRTL-5 cell proliferation and that somatostatin interferes with both signals. Each pathway activates different proteins required for G(1)/S progression. Thus, PKA is responsible for the TSH-induction of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA levels, RhoA activation, and down-regulation of p27(kip1). These correlated events are necessary for FRTL-5 cell proliferation after TSH stimulation. Moreover, TSH through PKA pathway increases cyclin-dependent kinase 2 levels, whereas PI 3-kinase signaling increases cyclin E levels. Together, both pathways finally converge, increasing the formation and activation of cyclin E x cyclin-dependent kinase 2 complexes and the phosphorylation of the retinoblastoma protein, two important steps in the transition from G(1) to S phase in growth-stimulated cells. Somatostatin exerts its antiproliferative effect inhibiting more upstream the TSH stimulation of PKA and PI 3-kinase, interfering with the TSH-mediated increases of intracellular cAMP levels by inactivation of adenylyl cyclase activity. Together, these results suggest the existence of a PKA-dependent pathway and a new PKA-independent PI 3-kinase pathway in the TSH/cAMP-mediated proliferation of FRTL-5 thyroid cells.

Somatostatin is expressed in FRTL-5 thyroid cells and prevents thyrotropin-mediated down-regulation of the cyclin-dependent kinase inhibitor p27kip1.

Using RT and amplification, we have detected specific RNA transcripts encoding somatostatin in FRTL-5 thyroid cells. This observation indicates that within the thyroid context, expression of somatostatin is not restricted to the parafollicular C cells. Transfection of FRTL-5 cells with constructs containing either the complete somatostatin gene promoter or deletions carrying the cAMP response element-binding site allowed us to demonstrate that transcription of the somatostatin gene is hormonally regulated by TSH. Blockage of somatostatin by specific antibodies resulted in an increased capacity of TSH-induced FRTL-5 cell-conditioned medium to promote cell proliferation, demonstrating that under physiological conditions, somatostatin exerts a cytostatic effect on FRTL-5 cells growth. Somatostatin treatment of FRTL-5 cells resulted in a growth retardation, caused by a dose-response delay in the G1 phase of the cell cycle. This effect appears to be mediated by the cyclin-dependent kinase inhibitor p27kip1, which is clearly down-regulated in FRTL-5 cells treated with TSH and whose expression is reestablished by somatostatin in a dose-dependent manner. Participation of somatostatin in the control of FRTL-5 cell proliferation is in agreement with the detection of specific somatostatin receptor type 2. Flow cytometric assays reveal that FRTL-5 cells transformed with the K-ras oncogene are still sensitive to somatostatin treatment, whereas fully neoplastic FRT cells no longer respond to this peptide. Taking together, the results demonstrate the participation of an autocrine loop in the control of thyroid cell proliferation, and the possibility that this mechanism could be altered in the process of thyroid carcinogenesis.

Growth regulation of rat thyrocytes (FRTL-5 cells) by the secreted ectodomain of beta-amyloid precursor-like proteins

The TSH-dependent expression of amyloid precursor-like proteins and the secretion of their ectodomain (sAPP) in rat thyroids coincide with increased rates of thyrocyte proliferation. To analyze whether the secretion of sAPP and the proliferation of thyrocytes are regulatorily linked, we employed [3H]thymidine or 5-bromo-2'-deoxyuridine assays and found that conditioned culture medium stimulated the proliferation of FRTL-5 cells depending on the content of sAPP. These observations prompted experiments with sAPP-derived peptides known to stimulate the growth of APP-deficient fibroblasts. Using autoradiography and radiochemical assays, we observed that an iodinated 19-mer sAPP peptide was bound specifically to the surface of FRTL-5 cells. Binding of this peptide was followed by a 2- to 8-fold increase in cell proliferation, which reached a plateau at 1 nM. This effect was significant only when cells were cultured in nonconfluent monolayers, and contact inhibition did not interfere. Our observations indicate that sAPP and sAPP-derived peptides increased the proportion of proliferation-competent FRTL-5 cells and suggest that sAPP may be a new member in the family of peptides involved in the growth regulation of thyrocytes.

Growth regulation of rat thyrocytes (FRTL-5 cells) by the secreted ectodomain of beta-amyloid precursor-like proteins.

The TSH-dependent expression of amyloid precursor-like proteins and the secretion of their ectodomain (sAPP) in rat thyroids coincide with increased rates of thyrocyte proliferation. To analyze whether the secretion of sAPP and the proliferation of thyrocytes are regulatorily linked, we employed [3H]thymidine or 5-bromo-2'-deoxyuridine assays and found that conditioned culture medium stimulated the proliferation of FRTL-5 cells depending on the content of sAPP. These observations prompted experiments with sAPP-derived peptides known to stimulate the growth of APP-deficient fibroblasts. Using autoradiography and radiochemical assays, we observed that an iodinated 19-mer sAPP peptide was bound specifically to the surface of FRTL-5 cells. Binding of this peptide was followed by a 2- to 8-fold increase in cell proliferation, which reached a plateau at 1 nM. This effect was significant only when cells were cultured in nonconfluent monolayers, and contact inhibition did not interfere. Our observations indicate that sAPP and sAPP-derived peptides increased the proportion of proliferation-competent FRTL-5 cells and suggest that sAPP may be a new member in the family of peptides involved in the growth regulation of thyrocytes.

The G2M arrest caused by iodide is unrelated to the effects of iodide at adenylate cyclase.

Previously, we have shown that the iodide was able to inhibit TSH induced thyrocyte proliferation by arresting the cell cycle at G0G1 and G2M, suggesting that the iodide may be exerting its effects through more than the TSH-adenylate cyclase-cAMP system. To confirm the effects of iodide on the adenylate cyclase (AC) system, forskolin- and dibutyryl-cyclic-AMP (dBcAMP)-stimulated FRTL5 thyroid cells were exposed to inhibitory concentrations of iodide and the resultant effects on the cell cycle were compared to the effects observed with TSH, using flow cytometric DNA analysis. Forskolin stimulated the proliferation of FRTL5 cells in a dose-dependent manner. Cell numbers rose from baseline by 169 +/- 4% to peak at 10 microM forskolin. Interestingly, 100 microM forskolin inhibited cell proliferation, causing cell numbers to fall by approximately 50%. Iodide inhibited forskolin-induced proliferation to baseline levels. However, the pattern of cell cycle perturbation was different to that with TSH-stimulated cells. There were no differences in the proportion of cells in G0G1 between forskolin alone and forskolin + NaI, while there was a marked fall in the proportion of cells in S phase, indicating possible partial arrest at G0G1. Furthermore, there was a marked accumulation of cells in G2M over and above that found with TSH + NaI, indicating arrest at G2M. dBcAMP maximally stimulated cell numbers to rise from baseline by 125% with 1 mM dBcAMP. Again, higher concentrations of the mitogen had an inhibitory effect on proliferation. The addition of NaI inhibited dBcAMP stimulated cell proliferation.

In the thyroid cells proliferation, differentiated and metabolic functions are under the control of different steps of the cyclic AMP cascade

In the course of studies to elucidate the complex network of interactions controlling FRTL5 cell proliferation, thyroid stimulating hormone (TSH)-independent mutants (M cells), have been obtained from FRTL5 cells by chemical mutagenesis. In the present studies, the role of TSH on the proliferation and on differentiated and metabolic functions in these mutant cells have been investigated and compared to their response to insulin-like growth factor I (IGF-I). The addition of IGF-I to M cells leads to normal stimulation of DNA synthesis. However, inspite of the fact that mutant cells display normal TSH receptors, TSH is unable to stimulate the proliferation of the M cells. Nevertheless, TSH is able to increase intracellular levels of cAMP leading to regulation of TSH function in the M cells. On the other hand, TSH does not influence iodide transport and actin filaments depolimerization in these cells. However, aminoacid transport, stimulated in wild-type FRTL5 cells by both TSH and IGFs, is under the control of IGFs but not of TSH in the mutant cells. Neither TSH or IGF-I modified the expression of C- fos proto-oncogene in the M cells, probably because of high constitutive expression. These data suggest that a crucial signalling step(s) required for TSH induced mitogenesis is impaired in the M cells, and that this signalling step is not required for IGF-I induced mitogenesis.

Effect of Interleukin-6 on Cell Proliferation of FRTL-5 Cells

We studied the effects of interleukin-6(IL-6) on DNA synthesis and cyclic AMP production in rat thyroid FRTL-5 cells. When cells were incubated with IL-6 in the presence or absence of IGF-I, cell proliferation was not observed. By contrast, IL-6 stimulated DNA synthesis in a dose dependent manner when TSH was added concomitantly. On the other hand, IL-6 did not modulate the cAMP accumulation in the presence or absence of TSH. These data demonstrate that, like IGF-I, IL-6 may be able to act as a growth factor through activation of a mitogenic signal transduction pathway different from A-kinase in FRTL-5 cells.

Effect of iodoarachidonates on thyroid FRTL-5 cells growth.

Excess iodide inhibits several thyroid parameters, by a putative organic iodocompound. Different iodolipids, including iodinated derivatives of arachidonic acid (IAs), are produced by rat, calf and pig thyroid. The action of two iodolactones, one bearing the iodine atom at the position 6 (IL-d) and the other at position 14 (IL-w) on growth of FRTL-5 cells was studied. KI, IL-w and IL-d exert a dose-related inhibition on FRTL-5 cell proliferation. The first two compounds caused inhibition at 1 microM while IL-d was effective at 10 microM. This inhibitory action of iodolactones (ILs) was not altered by 1 mM methyl-mercaptoimidazol (MMI), indicating that they exert their effect per se. The action of ILw on cell growth was reversible. The growth-stimulating effect of 10 microM forskolin was inhibited by IAs, showing that one possible site of action lies at the cAMP pathway. The present results give further support to our hypothesis about the role of IAs in thyroid growth autoregulation.

Effect of thyrotropin and cAMP on FRTL5 cell growth in a serum free medium

TSH-induced proliferation of FRTL5 cells was studied in a chemically serum-free, defined medium. FRTL5 cells incubated for several days in a medium lacking of serum and hormones were fully able to respond to TSH in terms of cAMP production and iodine uptake. In the same medium, TSH stimulated FRTL5 cell growth as assessed by thymidine incorporation, DNA content and cell count. In these experimental conditions the cellular doubling time was of 7.5 days as compared to 40 h when calf serum was present together with TSH. Cholera toxin, forskolin and (bu)2cAMP, substances able to selectively increase intracellular cAMP levels, were not as efficient as TSH in inducing FRTL5 cell growth. However, both TSH and (bu)2cAMP, in a similar way, induced c-myc gene expression and cellular progression through the prereplicative phase of cell division. These data demonstrate that cAMP-dependent mechanisms are only partially responsible for TSH-induced FRTL5 cell growth, thus suggesting a role of cAMP-independent mechanisms.

Transmembrane Na+/H+ exchange in the rat thyroid cell strain FRTL-5: a possible role in insulin-like growth factor-I-mediated proliferation

Using the fluorescent pH indicator 2'7'-bis(2-carboxyethyl)-5'-(6')-carboxyfluorescein to monitor intracellular pH (pHi), we have investigated whether transmembrane Na+/H+ exchange, as measured by experimental changes in pHi under bicarbonate-free incubation conditions, may be involved in the early growth-promoting actions of insulin-like growth factor-I (IGF-I) on the rat thyroid cell stain FRTL-5. In initial studies to characterize Na+/H+ exchange in FRTL-5 cell suspensions, the recovery of a resting pHi in acid-loaded cells was shown to be dependent upon the presence of extracellular Na+, was enhanced by the presence of the sodium ionophore monensin and was abolished by amiloride, an antagonist of Na+/H+ antiport activity. Unlike TSH, which was without effect on the pHi of FRTL-5 cells for up to 15 min after addition, IGF-I (1000 micrograms/l) caused a rapid and sustained increase within 3 min, which was abolished in medium in which Na+ had been replaced with an iso-osmotic level of choline chloride. The change in pHi in response to IGF-I was mimicked by phorbol 12-myristate 13-acetate (PMA; 100 nmol/l), an activator of thyroid cell proliferation. In the presence of TSH, exposure of cells to IGF-I or PMA had no additional effect on the cytoplasmic alkalinization induced by either of these two agonists alone. However, blockade of transmembrane Na+/H+ exchange with amiloride inhibited both the individual actions of IGF-I and PMA on [methyl-3H]thymidine incorporation, and the synergistic interaction between TSH and IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol esters have a dual action through protein kinase C in regulation of proliferation of FRTL-5 cells

In quiescent rat thyroid (FRTL-5) cells, phorbol-12-myristate-13-acetate (PMA) inhibited DNA synthesis induced by a combination of insulin and thyrotropin (TSH) or dibutyryl cyclic AMP (Bt 2cAMP). This inhibitory effect of PMA was observed even when PMA was added after addition of these growth factors, and was maximal when PMA was added 2–4 h after the growth factors (late in the G 1-phase of the cell cycle). On the other hand, PMA alone induced DNA synthesis and also enhanced that induced by Bt 2cAMP or insulin in these quiescent cells. 1-Oleoyl-2-acetylglycerol mimicked these effects of PMA, but 4α-phorbol-12,13-didecanoate had no effect. These data demonstrate that in FRTL-5 cells protein kinase C has a stimulatory effect on the G 0 to G 1 transition and an inhibitory effect on the G 1 to S transition in the cell cycle.

Role of the adenylate cyclase-cAMP system on TSH-stimulated thyroid cell growth.

TSH is a trophic factor for cultured rat thyroid cells (FRTL-5). In the present study we have investigated the mechanism by which TSH promotes cell growth and evaluated the possible role of the adenylate cyclase (AC)-cAMP system in this process. The mitogenic activity of several agents was evaluated by measuring their effect on cell number or 3H-thymidine incorporation into DNA. Forskolin and cholera toxin, two potent and specific activators of the AC, induced a dose dependent increase of 3H-thymidine incorporation. The maximal stimulation, observed at concentrations of 10 microM and 10 ng/ml, respectively, was beta 80% of that obtained with optimal concentrations of TSH. A similar effect was obtained with a Graves' IgG preparation (0.2 mg/ml) able to stimulate the thyroid AC or with 3-isobutyl-1-methyl-xanthine (IBMX, 0.5 mM), a phosphodiesterase inhibitor. 8-bromo cAMP (0.5 mM), a cAMP analog, also stimulated 3H-thymidine incorporation, and its potency was approximately 60% of that of TSH. Similar results were obtained when the mitogenic activity of these compounds was evaluated by cell number. Norepinephrine (NE, 10 microM), although devoid of AC stimulatory activity in these cells, also stimulated 3H-thymidine incorporation, but its potency was only 20-30% of that of TSH. Indomethacin (100 microM), an inhibitor of phospholipid and arachidonic acid metabolism, was able to inhibit the stimulatory effect of NE (84%), and to a lesser extent of TSH (63%) and cholera toxin, had minor effect on forskolin (24%), IBMX (16%) and Graves' IgG (8%), and no effect on 8-bromo cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)