Thyroid hormone receptors and 3,5,3'-triiodothyronine biological effects in FRTL5 thyroid follicular cells.

Specific thyroid hormone (T3) receptors are present in thyroid follicular cells, including the rat FRTL5 clonal line, but little is known about the effects of T3 on the growth and differentiated function of the thyroid. Unlike primary cultures of animal or human thyroid cells, FRTL5 do not secrete appreciable amounts of thyroid hormones. We now have studied the effects of T3 by itself and in combination with TSH and insulin-like growth factor-I (IGF-I) on [3H]thymidine incorporation into DNA, iodide uptake, and cAMP production in FRTL5. We also have investigated the expression of different c-erbA mRNAs in these cells. Specific binding of T3 to FRTL5 cell nuclei in intact cells occurred with a binding capacity of 0.1-0.15 ng T3/mg DNA and an apparent Kd of 0.4 nM. Using an RNase protection assay on total cellular FRTL5 RNA and specific cRNA probes, we demonstrated the presence of c-erbA alpha and -beta mRNAs, both encoding T3 receptors. Biological effects were assessed in serum-free medium or buffer containing 0.1% BSA after maintaining quiescent culture of cells for at least 5 days in hormone-free medium containing 5% calf serum. T3 alone stimulated a dose-dependent increase in [3H]thymidine incorporation that reached a plateau at 188% of the control value at 10 nM T3. At 10(-11) M TSH, T3 potentiated TSH-stimulated [3H]thymidine incorporation (2.2-fold), but at TSH concentrations greater than 5 x 10(-11) M, T3 had no effect or reduced the response to TSH. T3 potentiated the [3H]thymidine response to 2 and 10 ng/ml IGF-I by 1.5- to 1.7-fold. T3 alone had no effect on iodide uptake, but attenuated iodide uptake stimulated by TSH. T3 was more potent in inhibiting TSH-stimulated iodide uptake than in enhancing TSH-stimulated DNA synthesis. T3 did not affect either basal or TSH-stimulated cAMP accumulation. Thus, in FRTL5 thyroid follicular cells 1) T3 receptors are expressed, as measured by direct binding assays and by the expression of c-erbA mRNAs; and 2) T3 acts as a growth factor and weak antidifferentiation factor. We suggest that T3 may modulate the actions of TSH and growth factors in thyroid epithelium.     

Relationship between growth and function of human thyroid cells in culture

Subconfluent human thyroid cells in monolayer, isolated from thyrotoxic tissue or non-toxic goitres obtained at surgery, responded to the addition of epidermal growth factor (EGF) with an increase in cell growth as measured by increased incorporation of [3H]thymidine into trichloroacetic acid-precipitable material. The growth response to EGF was concentration-dependent and the characteristics of the responses were the same using EGF from murine or human sources. With concentrations which stimulated growth, EGF was found to inhibit human thyroid cell function as measured by the release of radioimmunoassayable tri-iodothyronine into the incubation medium. Thyrotrophin (TSH) was also found to stimulate human thyroid cell growth but at concentrations far lower than those used to stimulate thyroid cell function in this system. The effect of EGF on the differentiating action of TSH on human thyroid cells in culture was also investigated; the association of thyroid cells into two-dimensional follicular structures produced by the incubation of thyroid cells at a high cell density with TSH was found to be inhibited by the addition of EGF.     

Oestradiol is a potent mitogen and modulator of GnRH signalling in alphaT3-1 cells: are these effects causally related?

GnRH acts via phospholipase C (PLC) activating G-protein coupled receptors to stimulate secretion of gonadotrophins from gonadotrophs. These cells are also regulated by gonadal steroids, which act centrally to influence GnRH secretion, and peripherally to modulate GnRH action. We have shown that oestradiol can stimulate proliferation and modulate GnRH-stimulated [(3)H]inositol phosphate ([(3)H]IP(x)) accumulation (used as a measure of PLC activity) in a gonadotroph-derived cell line (alphaT3-1). Here we show that when alphaT3-1 cells were incubated in medium with 2% foetal calf serum (FCS), [(3)H]thymidine incorporation was not stimulated by oestradiol but was reduced to <2% of control by the oestrogen antagonist, raloxifene. The inhibitory effect of 10 or 1000 nM raloxifene was reversed competitively by oestradiol. A similar pattern of effects was seen when effects of oestradiol and raloxifene on the proportion of cells in the S-phase of the cell cycle (as measured by flow cytometry of propidium iodide-labelled cells) and on oestrogen receptor activity (as measured by trans-activation of the oestrogen-response elements in the vitellogenin promoter) were quantified. In addition, RT-PCR revealed expression of alpha and beta (but not beta2) subtypes of oestrogen receptors. Thus, oestrogen is an essential mitogen for alphaT3-1 cells, its mitogenic effect is oestrogen receptor mediated and is associated with a marked alteration of cell cycle distribution, and the full extent of these effects are best revealed in the presence of raloxifene. Using this strategy, we found that cells cultured for 4 days with 10 nM raloxifene expressed GnRH receptors (K(d) for (125)I-buserelin 4.33 nM) and that their activation by GnRH caused a concentration-dependent increase in [(3)H]IP(x) (in cells labelled with [(3)H]inositol) and inositol 1,4,5 trisphophate (in unlabelled cells). Addition of 10 nM oestradiol (to overcome receptor blockade by raloxifene) reduced GnRH receptor number by 31% but increased maximal effects on [(3)H]IP(x) and Ins(1,4,5)P(3) approximately 4-fold. The effects of oestradiol on GnRH receptor number and signalling were not, however, mimicked by culture for 2 days in medium with 10% FCS and the S-phase blocker, thymidine (15 mM). This treatment increased the proportion of cells in the S-phase 2- to 3-fold but did not alter GnRH receptor number or signalling. Other treatments which altered cell cycle transition (hydroxyurea, colcemid, methotrexate) also failed to alter GnRH receptor number or signalling and no correlation was seen between GnRH receptor number or GnRH-stimulated [(3)H]IP(x) accumulation and the proportion of cells in the S-phase or G2/M-phases of the cell cycle. Thus, oestradiol has pronounced effects on GnRH signalling, proliferation and cell cycle distribution in alphaT3-1 cells, but these trophic effects do not underlie the modulation of GnRH signalling.     

Thyrotropin modulates EGF receptor function in porcine thyroid follicle cells

Porcine thyroid follicle cells in monolayer cultures were shown to contain one single class of high-affinity EGF receptors with Kd = 4.5 X 10(-10) M and approximately 20 000-25 000 receptors per cell. Suspension cultures of aggregated follicle cells, exposed to TSH for 3 days, showed a 3-fold increase in [125I]EGF binding. Scatchard analysis demonstrated that this was due to an increase in receptor number. Other cAMP-elevating agents (cholera toxin, dibutyryl cAMP, forskolin) induced a similar effect. In suspension cultures, preincubation with TSH or cholera toxin for 2 days reduced the subsequent [3H]thymidine incorporation. This inhibition was overcome by a low concentration of EGF (0.1 ng/ml). At higher concentrations of EGF (1-10 ng/ml) the incorporation of [3H]thymidine was potentiated 2-3-fold in cultures preexposed to TSH or cholera toxin. The results demonstrate the presence of a high-affinity EGF receptor in porcine thyroid follicle cells. Receptor expression, as well as responsiveness to the mitogenic action of EGF, is modulated in vitro by TSH, through a cAMP-dependent process.     

A growth stimulatory effect of iodide is suggested by its effects on c-myc messenger ribonucleic acid levels, [3H]thymidine incorporation, and mitotic activity of porcine follicle cells in suspension culture

In the present study the effect of iodide on thyroid cell growth was investigated in primary suspension cultures of porcine thyroid cells capable of organifying iodide. The addition of a high dose of iodide (10(-4) M) to such cultures caused a marked increase in c-myc mRNA levels, [3H]thymidine incorporation, and mitotic activity. The incorporation of [3H]thymidine started 30-36 h after the addition of iodide. The stimulatory effect was abolished by a simultaneous incubation with methimazole. The concentration dependence of the iodide-induced stimulation of [3H]thymidine incorporation was similar to that of an inhibitory effect on adenylate cyclase activity. W-7, an inhibitor of calmodulin activity, as well as epinephrine, agents that reduce cAMP levels, also stimulated [3H]thymidine incorporation. Moreover, the stimulatory effect of iodide was reduced in the presence of forskolin. The results suggest that an organic form of iodine stimulates thyroid cell growth by reducing cAMP levels and demonstrate the presence of a growth stimulatory pathway in porcine thyroid cells that is independent of exogenous polypeptide growth factors or hormones.     

Antibody-dependent cell-mediated growth inhibition of rat thyroid cells, FRTL-5, in patients with autoimmune thyroid diseases

We studied antibody-dependent mononuclear cell-mediated growth inhibition of thyroid cells in 18 untreated patients with Graves' disease, 18 patients with chronic thyroiditis, and 15 normal subjects by measuring the ability of their sera to inhibit [3H]thymidine incorporation into DNA in a rat thyroid cell line, FRTL-5, in the presence of normal mononuclear cells. [3H]thymidine incorporation was significantly inhibited in the presence of sera from patients with Graves' disease and chronic thyroiditis (P less than 0.001), whereas it was not affected in normal subjects. A significant correlation was observed between the inhibition of [3H]thymidine incorporation and the titre of anti-microsomal antibodies (P less than 0.05). The inhibitory effect on [3H]thymidine incorporation was significantly abolished when serum pre-absorbed with human thyroid membranes was used (P less than 0.005). These inhibitory effects on [3H]thymidine incorporation significantly correlated with those obtained by using IgG fractions (P less than 0.01). These data indicate that antibody-dependent mononuclear cell-mediated growth inhibition may play a role in thyroid cell growth regulation in patients with autoimmune thyroid disease.     

Effects of epidermal growth factor on thyroglobulin and adenosine 3',5'-monophosphate production by cultured human thyrocytes

While several workers have identified epidermal growth factor (EGF) receptors on human thyroid membranes, very few reports have described EGF effects on intact human thyroid cells in primary culture, and these were short term studies indicating that EGF effects were primarily inhibitory [reduced iodide uptake and thyroglobulin (Tg), T4, and T3 release]. Paradoxically, in vivo EGF stimulates thyroid growth and increases colloid stores. In this study we examined the effects of EGF on cultured thyroid cells in regard to thymidine incorporation, Tg secretion, and cAMP production during a 12-day period. Addition of EGF (0-30 ng/mL) to medium for 6 or 12 days stimulated thymidine incorporation and enhanced Tg synthesis by thyroid cells. However, the profile of Tg release into medium was biphasic. Tg release was inhibited by EGF (0.1-10 ng/mL) during the first 3 days of culture, but the inhibitory effect disappeared by the sixth day, and EGF stimulated Tg release by day 12 and thereafter. EGF enhanced endogenous cAMP levels in thyroid cells, but did not augment TSH-stimulated increases in cAMP production. Our observations of EGF-stimulated growth and inhibited Tg secretion during short term culture are consistent with the findings of earlier studies with nonhuman thyrocytes. However, the later phase of enhanced cAMP levels with stimulation of Tg secretion indicates that EGF may have trophic effects on thyrocytes previously unrecognized because of the short term nature of the studies. These observations suggest an important role for EGF in maintenance of normal thyroid physiology.     

Effect of aging on EGF-induced proliferative response in primary cultured periportal and perivenous hepatocytes

BACKGROUND/AIMS: Aging relates to declined proliferative capacity of the liver, but the molecular mechanism is not well understood. We examined whether functional changes of epidermal growth factor (EGF) receptor (EGFR) are involved in age-related decline in EGF-induced DNA synthesis using hepatocytes isolated in periportal and perivenous regions of the liver, which differ in the proliferative capacity. METHODS: Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) in 7-, 30-, and 90-week-old rats were isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [methyl-(3)H]thymidine incorporation. EGFR binding affinity to EGF was analyzed by Scatchard analysis using [(125)I]EGF. EGFR dimerization and phosphorylation were determined by Western blot analysis. RESULTS: EGF-induced DNA synthesis was greater in PPH than in PVH from rats of 7 weeks, but the zonal difference disappeared with aging. [(125)I]EGF binding studies indicated that high-affinity EGFR in both subpopulations also disappeared with aging. Furthermore, EGF-induced dimerization in both subpopulations was down-regulated with aging, and the pattern of EGFR phosphorylation was parallel to that of dimerization. CONCLUSIONS: These data suggest that age-related decline in EGF-induced DNA synthesis of PPH and PVH is caused by down-regulation of EGFR dimerization through the decrease of high-affinity EGFR.     

Regulation of epidermal growth factor receptors by TSH, and effects of EGF, TSH and phorbol ester on DNA synthesis in cultured porcine thyroid cells

Using porcine thyroid cells of primary monolayer culture, this study was conducted to clarify the nature and characteristics of epidermal growth factor (EGF) receptors on porcine thyroid cells and also to investigate the effects of EGF, TSH and phorbol ester on DNA synthesis. Receptors for EGF on porcine thyroid cells exist in two forms which differ in both affinity and capacity. Scatchard analysis of saturation binding assay performed at 4 degrees C for 6h indicates that there is a high affinity class of receptors with low capacity (K1 = 4.70 +/- 0.90 X 10(-9)M and 8,600 +/- 1,200 sites/cell) and low affinity receptors with high capacity (K2 = 2.24 +/- 1.17 X 10(-7)M and 65,500 +/- 18,000 sites/cell) on the cells cultured for 4 days in the absence of TSH. When thyroid cells were cultured in the presence of various concentrations of TSH (0 approximately 50 mU/ml) and for various times (0 approximately 96 h) with TSH (10 mU/ml), specific EGF binding to the cells increased dose- and time-dependently. On TSH (10 mU/ml)-treated cells for 4 days, two kinds of EGF receptors, i.e. high affinity and low capacity (K1 = 5.39 +/- 1.75 X 10(-9) M and 17,200 +/- 2,500 sites/cell) and low affinity and high capacity (K2 = 1.70 +/- 1.40 X 10(-7)M and 76,300 +/- 17,900 sites/cells), were resolved. The results indicate that TSH can modulate EGF receptors by increasing the number of high affinity sites on porcine thyroid cells. Next, using [Me-3H] thymidine incorporation into TCA precipitable materials for 48h, we studied the biological effects of EGF, TSH and phorbol myristate acetate (one of the potent phorbol esters) on DNA synthesis. Both EGF and PMA can promote [Me-3H] thymidine incorporation. Maximal responses were obtained with EGF ranging from 10(-9) to 10(-7)M and with PMA from 10(-9) to 10(-7)M. In contrast, TSH inhibits [Me-3H] thymidine incorporation dose-dependently. Almost the same results were obtained by these agents on TSH (10 mU/ml)-treated cells for 4 days, but EGF stimulated cell growth at a lower concentration of 10(-11)M, which was possibly related to an increase of high affinity receptor numbers. The growth promoting effect of EGF and PMA in combination was additive, and TSH suppressed the cell growth in the concomitant presence of EGF and PMA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of thyroid autoantibodies on thyroid cellular growth in vitro

Porcine thyroid follicle cells, cultured in suspension, were employed to investigate the effects of immunoglobulin preparations from patients with colloid goitre, Graves' disease or Hashimoto's thyroiditis on thyroid growth in vitro. Epidermal growth factor (EGF, 19 ng/ml) was used as a reference for maximum growth stimulation and produced a 9-fold increase in [3H]thymidine incorporation. Immunoglobulins (1000 micrograms/ml) were found to increase [3H]thymidine incorporation compared to control: from 10 normal individuals 32 +/- 4% (mean +/- SEM, % of EGF response), from 10 patients with colloid goitre 26 +/- 4% (not significantly different from normal), from 10 patients with Graves' disease 19 +/- 3% (P less than 0.05) and from 15 patients with Hashimoto's thyroiditis 11 +/- 2% (P less than 0.001). No patient immunoglobulin preparation showed activity greater than that of normal individuals. The lower growth stimulatory activity in Graves' disease and Hashimoto's thyroiditis remained after heat inactivation of serum and is thought to reflect surface binding of thyroid autoantibodies.     

Repeatedly passed FRTL-5 rat thyroid cells can develop insulin and insulin-like growth factor-I-sensitive cyclooxygenase and prostaglandin E2 isomerase-like activities together with altered basal and thyrotropin-responsive thymidine incorporation into DNA

Repeatedly passed or aged rat FRTL-5 thyroid cells develop a high level of basal [3H]thymidine incorporation into DNA and a reduced response to TSH in medium containing 5% serum and insulin (5H medium). The basal [3H]thymidine incorporation into DNA of aged cells can exceed the TSH-induced increase in earlier passages of the same cell line (fresh cells) and the TSH response decreases from more than 10-fold above basal in fresh cells to less than 2-fold in aged cells. This change is not associated with a loss of the diploid karyotype, a change in basal cAMP levels, or a change in dependence on TSH for cell growth. Attenuation of the TSH response in the [3H]thymidine incorporation assay is more evident than the reduced effect of TSH on cAMP levels or iodide transport; moreover, the TSH effect on cAMP levels does not correlate with that on [3H] thymidine incorporation as a function of hormone concentration. The high basal activity in [3H]thymidine incorporation into DNA in aged cells is due to an increased responsiveness to insulin, insulin-like growth factor-I (IGF-I), or serum. Thus, removal of serum and insulin from the medium eliminates the high basal [3H]thymidine incorporation into DNA, and this activity is restored by insulin or IGF-I in a concentration-dependent manner. The increased responsiveness of aged cells to insulin or IGF-I is inhibited by indomethacin or hydrocortisone and is associated with insulin or IGF-I, but not TSH, stimulation of cyclooxygenase and prostaglandin E2 (PGE2) isomerase-like activity. Fresh cells, in contrast, require TSH plus insulin or IGF-I to increase these activities. Increased responsiveness of cyclooxygenase activity to insulin or IGF-I in aged cells reflects at least in part an increase in cyclooxygenase mRNA levels. We suggest that insulin/IGF-I stimulation of PGE2 production leads to the high basal thymidine incorporation into DNA in aged cells maintained in TSH-depleted (5H) medium; the reduced stimulation by TSH of cAMP content or iodide uptake may reflect PG inhibition (negative feedback regulation) of cAMP production.     

Inhibition of Replication in Functional Mouse Adrenal Tumor Cells by Adrenocorticotropic Hormone Mediated by Adenosine 3′:5′-Cyclic Monophosphate

Adrenocorticotropic hormone (ACTH) inhibited replication in functional adrenal tumor cells with a concomitant stimulation of steroidogenesis and a characteristic change of morphology from a flattened to a spherical type. [(3)H]Thymidine incorporation into DNA was inhibited by about 50% 6 hours after ACTH treatment. Both cyclic AMP and dibutyryl cyclic AMP inhibited [(3)H]thymidine incorporation and caused the characteristic morphological change noted with ACTH. The extent of stimulation of steroidogenesis and the amount of inhibition of [(3)H]thymidine incorporation in response to various doses of ACTH were closely related and both were in parallel with the concentration of cyclic AMP in the cells. Cyclic GMP and cyclic IMP did not inhibit [(3)H]thymidine incorporation significantly, and did not change the morphology of the cells. AMP inhibited [(3)H]thymidine incorporation into DNA and caused the characteristic morphological change. However, AMP did not increase the cyclic AMP content of the cells. CMP, GMP, and UMP showed a significant inhibition of [(3)H]thymidine incorporation into DNA, but the extent of the inhibition was much less than that with AMP. These nucleotides did not change the morphology of the cells.     

Regulation of amino acid uptake and deoxyribonucleic acid synthesis in isolated human fetal fibroblasts and myoblasts: effect of human placental lactogen, somatomedin-C, multiplication-stimulating activity, and insulin

We compared the abilities of human placental lactogen (hPL), somatomedin-C/insulin-like growth factor I (SM-C/IGF-I), multiplication-stimulating activity (MSA), and insulin to induce a rapid anabolic event, the uptake of the nonmetabolizable amino acid [3H]alpha-aminoisobutyric acid ([3H] AIB) or the more long term action of increasing [3H]thymidine incorporation, as a measure of DNA synthesis, in isolated human fetal fibroblasts and myoblasts. Myoblasts were derived from skeletal muscle and fibroblasts from skin explants removed from human fetuses delivered between 12 and 19 weeks gestation after prostaglandin-induced abortion. Each of the four peptides caused a dose-dependent increase in [3H]AIB uptake by both fibroblasts and myoblasts, with mean half-maximal concentrations (ED50) ranging from 0.9-1.9 nM. The concentration of each peptide required to stimulate [3H]thymidine uptake was significantly greater, with the exception of insulin, which was inactive. For myoblast cultures, the mean ED50 values were: hPL, 7.9 nM; SM-C/IGF-I, 2.0 nM; and MSA, 2.2 nM. For fibroblast cultures, the mean ED50 values were: hPL, 2.3 nM; SM-C/IGF-I, 3.3 nM; and MSA, 4.3 nM. Insulin did not stimulate [3H]thymidine incorporation into either cell type at concentrations up to 6.9 nM. Incubation in the presence of monoclonal antibody against SM-C/IGF-I abolished the ability of SM-C/IGF-I to stimulate either [3H]thymidine or [3H]AIB uptake into fetal fibroblasts. The antibody substantially inhibited the incorporation of [3H]thymidine by these cells in response to hPL, but was less effective in blocking hPL-stimulated [3H]AIB uptake. It did not inhibit the uptake of either radioisotope in response to MSA or [3H]AIB uptake in response to insulin. The actions of SM-C/IGF-I and hPL on thymidine incorporation were additive at submaximal concentrations, but not so at maximal individual concentrations. Their actions on AIB uptake were additive at both submaximal and maximal concentrations. The results suggest that hPL as well as the SMs may contribute to the growth stimulus in human fetal connective tissues. Since incubation with SM-C/IGF-I antibody reduced the mitogenic response of fetal cells to hPL, the actions on DNA synthesis may be partially mediated by local release of SM. However, the similar ED50 values with which these peptides stimulated [3H]AIB uptake during a short incubation, and their additive effects at maximal individual concentrations, suggest that hPL may also have direct actions.     

Activation of vascular smooth muscle cells by TNF and PDGF: overlapping and complementary signal transduction mechanisms

OBJECTIVE: Because tumor necrosis factor-alpha (TNF) has been implicated in the pathogenesis of vein graft neointimal hyperplasia, we sought to determine mechanisms by which TNF could induce proliferative and migratory responses in smooth muscle cells (SMCs). METHODS AND RESULTS: In rabbit jugulocarotid interposition vein grafts, SMCs expressed TNF as early as four days postoperatively. In rabbit aortic SMCs, TNF and platelet-derived growth factor (PDGF) elicited comparable migration (1.7-fold/basal), and their effects were partially additive. In contrast, while TNF failed to promote SMC [(3)H]thymidine incorporation alone, it doubled the [(3)H]thymidine incorporation observed with PDGF alone. To gain mechanistic insight into these phenomena, we found that TNF and PDGF each activated p38(mapk) equivalently in SMCs, but that PDGF was two to three times more efficacious than TNF in activating SMC extracellular signal-regulated kinases (ERK) 1 and 2 and phosphoinositide 3-kinase. However, only TNF activated NF kappa B. SMC [(3)H]thymidine incorporation that depended on TNF, but not PDGF, was abolished by overexpression of a dominant-negative I kappa B alpha mutant. Inhibition of ERK activation by U0126 reduced SMC migration stimulated only by PDGF (by 35%, P<0.05), but not by TNF. Inhibition of phosphoinositide 3-kinase by LY294002, however, significantly reduced both TNF- and PDGF-stimulated chemotaxis (by 38-54%, P<0.05). In contrast, both U0126 and LY294002 abolished SMC [(3)H]thymidine incorporation induced by either TNF, PDGF, or both agonists. CONCLUSIONS: In primary rabbit SMCs, TNF promotes migration and mitogenesis through signaling mechanisms that are both distinct from and overlapping with those employed by PDGF. TNF-induced SMC mitogenesis requires complementary co-stimulation with other growth factors.     

Increased thymidine kinase activity in human thyroid toxic adenomas: effects of exposure to epidermal growth factor in vitro

The activity of thymidine kinase (TK-EC 2.7.1.21)--an enzyme functioning as a part of the pyrimidine salvage pathway of DNA synthesis--is closely related to growth processes. The aim of the study was to measure TK activity in homogenates of human thyroid tissue of the following types: non-toxic nodular goiter (NTNG)--macroscopically unchanged tissue, non-toxic adenoma (NTA), and toxic adenoma (TA) (obtained from patients, who--before the surgery--had been treated with thyrostatic drugs for thyrotoxicosis). Thyroid tissue was obtained from female patients subjected to subtotal thyroidectomy at the Department of Endocrine Surgery, Medical University of Lód?. Thyroid homogenates were incubated in the presence of epidermal growth factor (EGF), used in five different concentrations (0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml). TK activity was estimated by chromatographic measurements of the amount of the main reaction product--deoxythymidine monophosphate. Results: 1) We did not observe any significant difference between TK activity in the homogenates of the thyroid tissue collected from NTNG and NTA; TK activity was clearly higher in the homogenates of adenomatous tissue, collected from the patients with TA; 2) EGF increased TK activity in the homogenates of the macroscopically unchanged tissue, collected--during surgery--from the patients with NTNG, as well as in homogenates of thyroid tissue from NTA; 3) In case of hyperactive thyroid tissue, obtained from TA, EGF tended to increase TK activity, however, without any statistical differences. Our results confirm TK increased activity in hyperactive thyroid tissue. At the same time, the obtained data suggest a certain role of EGF in goiter formation in humans.     

Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells

The effects of Escherichia coli heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [(3)H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [(3)H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.     

Blastic Transformation of Mouse Spleen Lymphocytes by a Water-Soluble Mitogen Extracted from Nocardia

A water-soluble extract of Nocardia markedly increased in vitro [(3)H]thymidine incorporation by mouse spleen lymphocytes. The blastogenic activity of the extract and lipopolysaccharide was studied comparatively on various mouse lymphocyte subpopulations. The data obtained by [(3)H]thymidine incorporation and by electron microscopy have demonstrated that this preparation stimulates selectively mouse bone-marrow-derived cortisone-sensitive lymphocytes. This stimulation is related neither to a natural infection of mice with Nocardia organisms nor to the presence in Nocardia water-soluble mitogen of a lipopolysaccharide contaminant or of a lipopolysaccharide-like material.     

Development of potent gastrin-releasing peptide antagonists having a D-Pro-psi(CH2NH)-Phe-NH2 C terminus.

Gastrin-releasing peptide (GRP) is a 27-amino acid neuroendocrine hormone that may play a role in the pathophysiology of small cell lung carcinoma. GRP and bombesin, a structurally related peptide, stimulate the growth of some cultured cell types. C-terminal GRP peptide analogs were developed that inhibited 6 nM bombesin-induced [3H]thymidine incorporation into quiescent murine Swiss 3T3 cells, which routinely produced a 6-fold stimulation over the basal extent of incorporation. The peptides were also analyzed for their capacity to inhibit the binding of 50 pM 125I-labeled GRP to Swiss 3T3 cells. The combination of two chemical modifications, each antagonistic in itself, led to the creation of antagonists with orders of magnitude greater potency than either modification alone. (i) Antagonist analogs of the form -Leu26-psi(CH2NH)-Xaa27-NH2 [where Xaa is Leu, norleucine (Nle), or Phe; residues numbered after GRP], similar to those introduced by Coy and coworkers [for review, see Jensen, R. T. & Coy, D. H. (1991) Trends Pharmacol. Sci. 12, 13-19], were found to have nanomolar potencies. (ii) We found that an octapeptide C-terminal GRP analog having D-Pro adjacent to the C-terminal amino acid amide was antagonistic, with a potency of 40 nM. By combining both modifications, specific analogs were found with potencies > 1000-fold greater than our lead structure--[(4'-hydroxy)-3-phenylpropanoyl]-Pro-Arg-Gly-Asn-His-Tr p-Ala-Val - Gly-His-Leu-psi(CH2NH)-Nle-NH2--and greater than any antagonist previously reported. The analogs [(4'-hydroxy)-3-phenylpropanoyl]-His-Trp-Ala-Val-D-Ala-His-D-Pro- psi(CH2NH)-Phe-NH2 and 1-naphthoyl-His-Trp-Ala-Val-D-Ala-His-D-Pro-psi(CH2NH)-Phe-NH2 antagonized [3H]thymidine incorporation with IC50 values of approximately 0.3 nM and inhibited the binding of 125I-labeled GRP with IC50 values of approximately 1 pM. These peptides may be of use in the study of the physiology of GRP.     

Effects of triiodothyronine (T3) and identification of specific nuclear T3-binding sites in cultured human fetal epiphyseal chondrocytes.

The effects of T3 on cultured human fetal epiphyseal chondrocytes were assessed by studying its effects on DNA synthesis and alkaline phosphatase activity. DNA synthesis was evaluated as follows: after 48-h incubation in Ham's F-12 serum-free medium, cultured chondrocytes were incubated with or without T3 (0.1-100 nM) in MCDB-104 serum-free medium for different periods of time (2-10 days), with the addition of [3H]thymidine (5 microCi/mL) for the last 24 h. Confluent cultured chondrocytes in 25-cm2 tissue culture flasks were incubated in Ham's F-12 serum-free medium for up to 9 days with or without T3 (0.1-100 nM); the cellular cytoplasmic fraction was obtained, and alkaline phosphatase activity was evaluated using paranitrophenylphosphate as a substrate. No significant effects of T3 (0.1-100 nM) on DNA-[3H]thymidine incorporation were observed in any experiment (n = 17) for any gestational age (12-39 weeks) or for any incubation period studied (2-10 days). However, a significant (P less than 0.025 or more) stimulatory effect of T3 (0.1-100 nM) on alkaline phosphatase activity was observed after 9 days of incubation. This effect was highest for 5 nM T3 and was present in cultured chondrocytes from human fetuses of all ages studied (13-40 weeks). Cultured human fetal epiphyseal chondrocytes from human fetuses 12-40 weeks old (n = 8) showed specific nuclear binding sites for T3. The binding capacity was 27.14 +/- 2.84 fmol/100 micrograms DNA, and the Kd was 0.66 +/- 0.14 x 0.1 nM (mean +/- SEM), with no significant differences among fetal ages. In conclusion, our results show that T3 elicits a biological response in cultured human fetal epiphyseal chondrocytes and has specific nuclear binding sites. Since alkaline phosphatase is closely related to the mineralization of epiphyseal cartilage, these results suggest that thyroid hormones could regulate this process.