Characterisation of the cyclic AMP response to thyrotrophin in monolayer cultures of normal human thyroid cells

1981 ◽  
Vol 98 (3) ◽  
pp. 370-376 ◽  
Author(s):  
Stephen P. Bidey ◽  
Nicholas J. Marshall ◽  
Roger P. Ekins
Keyword(s):  
Cyclic Amp ◽  
Dose Range ◽  
Thyroid Tissue ◽  
Human Thyroid ◽  
Thyroid Cells ◽  
Monolayer Cultures ◽  
Normal Human ◽  
Stimulation Of ◽  
In Cells

Abstract. The cyclic AMP response to thyrotrophin (TSH) has been investigated in cells prepared from human thyroid tissue obtained during surgery for sub-total laryngectomy, and maintained under in vitro conditions as primary monolayer cultures. When cells were incubated with 1.0 mU TSH/ml, a maximal level of intracellular cyclic AMP was reached after 20 min of incubation in the presence of 0.5 mm 3-isobutyl-1-methyl xanthine (MIX). This level of cyclic AMP was sustained for at least 2 h. Half-maximal stimulation of cyclic AMP was produced by TSH doses of between 1 and 5 mU/ml. In a study of a series of eight groups of monolayer cultures, each derived from a single, different thyroid gland, the mean stimulation of cyclic AMP given by 50 mU TSH/ml was 37.8-fold greater than in non-stimulated cell monolayers. Significant stimulation to 50 μU TSH/ml was observed in some monolayers and the precision of measurement of TSH was better than 15% over the TSH dose range 0.2–1.0 mU/ml. The magnitude of the cyclic AMP response to TSH was unaffected by the presence in the incubation medium of 20% (v/v) normal human serum. A cyclic AMP response to TSH was still demonstrable in cells that had been maintained for a period of 22 days in monolayer culture, although the response was reduced in comparison with that given by 4–5 day old cultures.

IN-VITRO STUDIES OF NORMAL HUMAN THYROID CELLS: RESPONSES TO THYROTROPHIN AND DIBUTYRYL CYCLIC AMP

1977 ◽  
Vol 72 (1) ◽  
pp. 87-96 ◽  
Author(s):  
S. P. BIDEY ◽  
P. MARSDEN ◽  
J. ANDERSON ◽  
C. G. McKERRON ◽  
H. BERRY
Keyword(s):  
Cyclic Amp ◽  
Thyroid Tissue ◽  
Three Dimensional ◽  
Human Thyroid ◽  
Dibutyryl Cyclic Amp ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Normal Human

SUMMARY Follicular cells isolated from normal human thyroid tissue have been cultured for up to 140 h with bovine thyrotrophin (TSH) or dibutyryl cyclic AMP (DBcAMP). Both compounds induced marked reorganization of the cells into three-dimensional follicular structures, whilst non-supplemented cells assumed a monolayer form. Cultures treated initially with TSH or DBcAMP showed a greater iodide uptake capacity, in comparison with unsupplemented cultures, in which iodide uptake was markedly diminished after 24 h. The release of tri-iodothyronine (T3) and thyroxine (T4) into the medium was determined by radioimmunoassay. Both TSH- and DBcAMP-treated cells showed a significant increase in iodothyronine output compared with unsupplemented control cells. In contrast to the 'classical' TSH-induced depression of the T4:T3 ratio in vivo, an increase in the ratio was observed for both TSH- and DBcAMP-supplemented cells in vitro. The ratio was also significantly greater after TSH than after DBcAMP, and possible implications of this finding are discussed.

Cyclic AMP release from normal human thyroid slices in response to thyrotrophin

1980 ◽  
Vol 95 (3) ◽  
pp. 335-340 ◽  
Author(s):  
Stephen P. Bidey ◽  
Nicholas J. Marshall ◽  
Roger P. Ekins
Keyword(s):  
Cyclic Amp ◽  
Incubation Time ◽  
Thyroid Tissue ◽  
Human Thyroid ◽  
Dose Dependency ◽  
Incubation Periods ◽  
In Vitro Exposure ◽  
Normal Human ◽  
Stimulation Of

Abstract. Slice preparations of normal human thyroid tissue were incubated in vitro with TSH. The cyclic AMP contents of slices were determined at intervals up to 120 min, and cyclic AMP in the incubation medium was also estimated for each incubation period. Slice cyclic AMP levels were related both to incubation time and TSH dose. In response to 10 mU TSH/ml, slice cyclic AMP levels were maximal within 60 min, and were not significantly changed at 120 min. Cyclic AMP was detectable in the medium within 10 min of slice exposure to TSH, and increased throughout the initial 60 min of incubation. Cyclic AMP release during this period was dependent on both TSH dose and incubation time. Between 60–120 min, however, cyclic AMP release partially lost its TSH dose-dependency, and 0.5–5.0 mU TSH/ml were equipotent with respect to the final medium cyclic AMP level attained. Slices incubated without TSH released only small amounts of cyclic AMP, and maximal levels were attained within 20 min. In contrast to the adenylate cyclase response of thyroid membrane preparations, which was stimulated by NaF, suggesting that cyclic AMP release was not a result of the stimulation of damaged cells. These findings demonstrate the importance of cyclic AMP release from human thyroid slices, following in vitro exposure to TSH, and suggest that, after incubation periods such as are used for the functional biodetection of thyroid stimulators, the magnitude of cyclic AMP release may be of quantitative significance.

A characterisation of cyclic AMP release from monolayer cultures of normal human thyroid cells

1982 ◽  
Vol 101 (3) ◽  
pp. 359-364 ◽  
Author(s):  
Stephen P. Bidey ◽  
Nicholas J. Marshall ◽  
Roger P. Ekins
Keyword(s):  
Cyclic Amp ◽  
Incubation Medium ◽  
Detection System ◽  
Human Thyroid ◽  
Thyroid Cells ◽  
Monolayer Cultures ◽  
A Cell ◽  
Normal Human ◽  
Primary Monolayer ◽  
Primary Monolayer Cultures

Abstract. The thyrotrophin (TSH)-dependent and time-related release of cyclic AMP has been characterised in primary monolayer cultures of normal human thyroid cells. Accumulation of cyclic AMP within the incubation medium was detectable within 1 h of exposure of cultures to 5 mU TSH/ml, and increased throughout of subsequent 15 h incubation period, final levels attained being consistently in excess of the corresponding intracellular cyclic AMP levels. Accumulation of cyclic AMP in the incubation medium was dependent on TSH dose, for both short (1 h) and prolonged (16 h) incubations. Moreover, after incubation for 16 h, cyclic AMP levels in the incubation medium were significantly (P < 0.01) in excess of intracellular levels for each dose of TSH tested above 0.2 mU/ml. In the absence of TSH, accumulation of cyclic AMP in the incubation medium remained low, after both 1 h and 16 h incubation periods. A consideration of these observations suggests that a bioassay based upon the cyclic AMP content of incubation medium samples should provide a more precise detection system for thyroid stimulators than those measuring the intracellular cyclic AMP response, and this has been demonstrated for a cell preparation in which the intracellular response to TSH was minimal.

Characteristics of thyrotrophin-stimulated cyclic AMP production in cultured human thyroid cells from thyrotoxic tissue and non-toxic goitres

1982 ◽  
Vol 95 (2) ◽  
pp. 237-244 ◽  
Author(s):  
C. A. Ollis ◽  
D. S. Munro ◽  
S. Tomlinson
Keyword(s):  
Cyclic Amp ◽  
Thyroid Tissue ◽  
Human Thyroid ◽  
Maximal Response ◽  
Cell Passage ◽  
Thyroid Cells ◽  
Response Characteristics ◽  
In Cells

We have described a system for the maintenance in culture of isolated human thyroid cells from both thyrotoxic tissue and non-toxic goitres. The cells isolated from the two thyroid tissue types showed similar cyclic AMP response characteristics to TSH with large increases in intracellular and extracellular cyclic AMP after 20-min incubations. Maximal responses were obtained with 50 mu. TSH/ml and half-maximal responses at 1·0 mu. TSH/ml. With cell passage the cyclic AMP responses to TSH decreased in magnitude and sensitivity. As with other thyroid cultures, growth of the cells with TSH induced arrangement into follicular structures, whereas cells grown in the absence of TSH remained as a monolayer. Basal intracellular cyclic AMP levels were increased in a dose-related fashion in cells grown in the presence of graded concentrations of TSH and the maximal response to further additions of TSH was not greater than in control cultures.

INHIBITION BY NORMAL IMMUNOGLOBULINS OF THYROTROPHIN-STIMULATED PRODUCTION OF CYCLIC AMP IN SLICES OF NORMAL HUMAN THYROID

1980 ◽  
Vol 87 (2) ◽  
pp. 271-277 ◽  
Author(s):  
S. P. BIDEY ◽  
N. J. MARSHALL ◽  
R. P. EKINS
Keyword(s):  
Cyclic Amp ◽  
Immunoglobulin G ◽  
Thyroid Tissue ◽  
Human Thyroid ◽  
Human Thyroid Tissue ◽  
Thyroid Stimulating Antibodies ◽  
Cyclic Amp Accumulation ◽  
Normal Human ◽  
Stimulation Of

Slice preparations of normal human thyroid tissue have been used to investigate the effect of normal immunoglobulin G (IgG) on thyrotrophin (TSH)-induced accumulation of cyclic AMP. Incubation of slices in the presence of both TSH and normal IgG for 20 min reduced the stimulation of cyclic AMP accumulation elicited by TSH alone by approximately 30%. However, preincubation of slices with IgG for 100 min before addition of TSH virtually abolished the response to TSH. The latter effect of normal IgG was reversible, and removal of IgG before exposure to TSH allowed an unimpaired cyclic AMP response to TSH. The implications of these observations with respect to the application of this system to the functional bio-detection of thyroid-stimulating antibodies in IgG fractions from thyrotoxic sera are discussed.

Evaluation of the rat thyroid cell strain FRTL-5 as an in-vitro bioassay system for thyrotrophin

1984 ◽  
Vol 101 (3) ◽  
pp. 269-NP ◽  
Author(s):  
S. P. Bidey ◽  
L. Chiovato ◽  
A. Day ◽  
M. Turmaine ◽  
R. P. Gould ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Tissue Slices ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Rat Thyroid ◽  
Bovine Tsh ◽  
Stimulation Of

ABSTRACT The cyclic AMP response to bovine TSH was characterized in a strain of rat thyroid follicular cells (FRTL-5) maintained in continuous culture. Significant stimulation of intracellular cyclic AMP was attained at a TSH dose of 5 μu./ml. Cyclic AMP accumulation continued to increase, at higher TSH doses, with no evidence for attainment of a maximum level at the highest dose tested (5 mu./ml). The precision of TSH measurement was better than 10% over the range 50–5000 μu./ml, comparing favourably with that observed with analogous assays based on human cells, tissue slices or membrane preparations. Using sequential subcultures of FRTL-5 cells, the between-assay variation in response to a single dose of a standard preparation of bovine TSH (53/11; 370 μu./ml) was of the order of 20% which compared favourably with the between-assay variation observed with different cultures of human thyroid cells. Prolongation of the incubation of FRTL-5 cells with TSH to 3 h revealed a progressive increase in the extracellular accumulation of cyclic AMP. Addition of TSH to resting FRTL-5 cells resulted in a stimulation of inorganic iodide uptake with pronounced bell-shaped dose–response characteristics. Thus a maximum uptake was observed at a TSH dose of 100 μu./ml with a significant reduction at higher doses. Acute stimulation of cells with TSH (100 μu./ml) resulted in a rapid and marked alteration in cell morphology, with evidence of cellular retraction and surface ruffling. J. Endocr. (1984) 101, 269–276

scholarly journals Dose-Dependent Generation of RET/PTC in Human Thyroid Cells after in Vitro Exposure to γ-Radiation: A Model of Carcinogenic Chromosomal Rearrangement Induced by Ionizing Radiation

2005 ◽  
Vol 90 (4) ◽  
pp. 2364-2369 ◽  
Author(s):  
Christy M. Caudill ◽  
Zhaowen Zhu ◽  
Raffaele Ciampi ◽  
James R. Stringer ◽  
Yuri E. Nikiforov
Keyword(s):  
Ionizing Radiation ◽  
Average Rate ◽  
Human Thyroid ◽  
Human Populations ◽  
Thyroid Cells ◽  
Genetic Event ◽  
Γ Radiation ◽  
Dose Dependent ◽  
In Cells

Abstract Ionizing radiation is a well-known risk factor for thyroid cancer in human populations. Chromosomal rearrangements involving the RET gene, known as RET/PTC, are prevalent in thyroid papillary carcinomas from patients with radiation history. We studied the generation of RET/PTC in HTori-3 immortalized human thyroid cells exposed to a range of doses of γ-radiation and harvested 2, 5–6, and 9 d later. RET/PTC1 and RET/PTC3 were detected by RT-PCR followed by Southern blotting and hybridization with internal oligonucleotide probes. No RET/PTC was found in cells harvested 2 and 5–6 d after irradiation, whereas 59 RET/PTC events were detected in cells collected 9 d after exposure. The average rate of RET/PTC induction was 0.1 × 10−6 after exposure to 0.1 Gy, 1.6 × 10−6 after 1 Gy, 3.0 × 10−6 after 5 Gy, and 0.9 × 10−6 after 10 Gy. When adjusted for cell survival, the rate after 10 Gy was comparable with those after 5 Gy. RET/PTC1 was more common than RET/PTC3 after each dose, comprising 80% of all rearrangements. In this study, we demonstrate a dose-dependent induction of RET/PTC rearrangements in human thyroid cells after exposure to 0.1–10 Gy γ-radiation. This provides additional evidence for a direct link between this genetic event and radiation exposure and offers a powerful experimental system for studying radiation-induced carcinogenesis in the thyroid gland.

scholarly journals Induction of follicle formation in long-term cultured normal human thyroid cells treated with thyrotropin stimulates iodide uptake but not sodium/iodide symporter messenger RNA and protein expression

2000 ◽  
Vol 167 (1) ◽  
pp. 125-135 ◽  
Author(s):  
T Kogai ◽  
F Curcio ◽  
S Hyman ◽  
EM Cornford ◽  
GA Brent ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Sodium Iodide ◽  
Human Thyroid ◽  
Sodium Iodide Symporter ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Protein Levels ◽  
Normal Human ◽  
Tsh Stimulation ◽  
Stimulation Of

Iodide uptake by the sodium/iodide symporter (NIS) in thyrocytes is essential for thyroid hormone production. Reduced NIS activity has been reported in thyroid diseases, including thyroid cancer and congenital hypothyroidism. The study of iodide uptake in thyrocytes has been limited by the availability of appropriate in vitro models. A new culture technique was recently developed that allows normal human thyroid primary culture cells to grow as monolayer cells and express differentiated functions for more than 3 months. We used this technique to study the effect of follicle formation and TSH on iodide uptake in these cells. Iodide uptake by the cells grown in monolayer was very low. Follicle formation was induced from monolayer cells, and electron micrographs demonstrated cell polarity in the follicles. No significant increase in iodide uptake was observed after TSH treatment of cells in monolayer or when follicle formation was induced without TSH. TSH stimulation of follicles, however, significantly increased iodide uptake ( approximately 4. 4-fold; P<0.001). Compared with iodide uptake in monolayers, the combination of follicle formation and TSH treatment stimulated iodide uptake synergistically to 12.0-fold (P<0.001). NIS messenger RNA (mRNA) and protein levels were almost the same in both monolayer cells and follicles. TSH treatment of monolayers and follicles produced significant (P<0.05) stimulation of mRNA ( approximately 4. 8- and approximately 4.3-fold respectively) and protein ( approximately 6.8- and 4.9-fold respectively). TSH stimulated NIS protein levels in both monolayer and follicles, however, stimulation of functional iodide uptake was only seen with TSH stimulation of follicles. The function of NIS may involve post-transcriptional events, such as intracellular sorting, membrane localization of NIS or another NIS regulatory factor. Polarized functions, such as iodide efflux into follicular lumina, may also contribute to the increased iodide concentration after follicle formation.

Responses In Vitro of Human Thyroid Cells to Thyrotrophin and Dibutyryl Cyclic amp

1976 ◽  
Vol 50 (2) ◽  
pp. 29P-30P
Author(s):  
S. P. Bidey ◽  
P. Marsden ◽  
C. G. McKerron ◽  
J. Anderson
Keyword(s):  
Cyclic Amp ◽  
Human Thyroid ◽  
Dibutyryl Cyclic Amp ◽  
Thyroid Cells
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