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Sex steroid-binding activities have been identified by several authors in normal and pathological thyroids and the expression of the canonic androgen receptor (AR) has recently been demonstrated in human thyroid follicular cells. In order to assess what influence, if any, androgen exposure has on thyroid cell growth, the effect of dihydrotestosterone (DHT) on [3H]thymidine (thy) incorporation and cell proliferation was investigated in thyroid follicular cells in vitro. In a primary culture of goitrous cells, DHT induced a significant reduction of [3H]thy incorporation at concentrations ranging from 10(-12) to 10(-8) M, with a more pronounced effect at 10(-9) M. At this concentration, the inhibitory effect was evident after both 24 and 48 h of treatment and in various types of primary thyroid cell cultures. In goitrous cells, the DHT-induced decrease of [3H]thy was associated with a reduction of expression of the proliferation-associated nuclear Ki-67 antigen, a protein commonly used to assess cell growth fraction. In TPC cells, an AR-positive thyroid papillary carcinoma cell line, DHT at concentrations between 10(-12) and 10(-8) M significantly decreased the growth rate. DHT (10(-9) M) produced an approximately 50-60% inhibition of cell proliferation and the antiandrogen cyproterone acetate was capable of reversing such effects. The DHT-induced reduction of TPC cell proliferation was associated with a significant reduction of c-myc RNA levels. Thyroperoxidase mRNA levels and thyroglobulin production were not reduced by androgen in primary cultures of goitrous cells. In conclusion, our results indicated that androgens may have a role in this gland by reducing the proliferation, but not the function, of follicular cells.

Androgen-binding activity has been identified in normal and pathological thyroids, but evidence for the expression of the canonic androgen receptor (AR) in the thyroid has not been provided so far. In this study we have used reverse transcription (RT)-PCR to examine RNA expression of the canonic AR gene in human thyroid tissues, in primary cultures of human thyrocytes and in a variety of neoplastic thyroid cell lines (NPA, TPC and WRO). An AR cDNA fragment with the expected size of 262 bp was detected in normal tissues and cultured thyrocytes as well as in neoplastic cell lines, demonstrating that the gene for AR is indeed expressed in thyroid follicular cells. Immunocytochemical analysis revealed the presence of the AR protein in cancer cell lines and androgen treatment increased nuclear positivity to AR. In a survey of 35 thyroid tissues AR cDNA was detected in all the non-neoplastic samples (6 normal and 3 goitrous) and in 19 of 26 neoplastic samples. AR cDNA was not detected in 4 of the 9 follicular adenomas and in 3 of the 12 papillary carcinomas. AR was revealed by immunohistochemistry in 1 of 2 normal thyroids, in 1 goiter and in 1 of 2 neoplastic thyroids. These findings show the presence of the canonic AR in the human thyroid.

The expression of all-trans-retinoic acid receptor (RAR) RNA was investigated by Northern blot and Reverse Transcription-Polymerase Chain Reaction in tissues and primary cultures of human thyrocytes. In normal and adenomatous samples the RAR alpha RNA was expressed, whereas the expression of RAR beta and gamma was undetectable. In carcinoma samples RAR alpha RNA expression could decline, whereas the RAR beta RNA expression could become detectable. TSH and retinoic acid did not significantly modify RAR alpha mRNA levels, whereas RA caused a significant decrease in basal and TSH-induced thyroid peroxidase (TPO) mRNA levels, and a decrease in DNA synthesis. These results demonstrate that RAR alpha gene is predominantly expressed in human thyrocytes, and suggest a molecular link between this gene and the negative regulation by RA of proliferation and function of follicular cells.

In order to clarify the effect of retinoids on thyroid cell growth and function, the presence of retinoic acid receptors (RARs) and the action of retinoic acid (RA) on DNA synthesis and on thyroid peroxidase (TPO) and thyroglobulin (TGB) mRNA expression were investigated in primary cultures of human thyroid follicular cells. A time and dose-dependent reduction in 3H-thymidine (3H-thy) incorporation was found in cells exposed for 48 h to all-trans-RA up to 1 microM. A cytotoxic effect was found only with the higher dose of 50 microM. The RA-induced decrease of 3H-thy incorporation was reflected by parallel change in DNA content of cell monolayers. The inhibitory effect of 1 microM RA on 3H-thy incorporation ranged from 28.5 +/- 4.6% in normal cells to 42 +/- 3.2% in adenomatous cells. In addition, 1 microM RA significantly reduced basal and TSH-induced TPO mRNA levels in normal, goitrous and adenomatous cells, but did not alter TGB mRNA levels. Furthermore, in these cells the study of RAR alpha and beta mRNA showed the presence of two major RAR alpha mRNA transcripts of approximately 3.5 and 2.8 Kb in size, whereas RAR beta mRNA was undetectable. Overall, our data indicate that RAR alpha gene is expressed in human thyrocytes and that RA may be involved in the regulation of the human thyroid by reducing proliferation and function of follicular cells.

In order to demonstrate that Androgen binding activity in thyroid is caused by the canonic Androgen Receptor (AR), member of steroid receptor family, we studied the presence of AR mRNA in human thyroid tissues and primary cultured cells. Here we report a polymerase chain reaction protocol (RT-PCR) that we have designated to investigate the presence of AR mRNA in human cells. AR cDNA was synthesized and amplified with primers specific for C-terminal sequence of the protein. We demonstrated that AR gene expression i) is present in thyroid samples studied and ii) in a primary culture of follicular adenoma where it seems to be modulated by steroid hormones.

The action of somatostatin (SRIH) on 3H-thymidine (thy) incorporation and on c-myc and thyroglobulin RNA levels in a suspension of follicles from normal and goitrous human thyroid was examined. SRIH, at 10(-7) M concentration, inhibited basal thy incorporation (maximally by 4 h lasting for up 24 h), which effect was greater in goiter than in normal thyroid and was also detected in growing adherent epithelial cells. Moreover, in a follicle suspension SRIH prevented TSH-stimulated thy incorporation, both in normal and in goitrous thyroid. Basal expression of c-myc RNA was not affected by SRIH in either tissue, whereas the TSH-stimulated c-myc RNA level was significantly reduced in goiter. No effect of SRIH was observed on basal or TSH-stimulated thyroglobulin RNA levels. SRIH did not alter basal cAMP concentrations in normal or goitrous follicles, but it significantly reduced TSH-stimulated cAMP accumulation both in normal thyroid and in goiter. Overall, our data indicate a direct inhibitory action of SRIH on growth, but not on differentiation, of human thyroid, probably by a mechanism not entirely cAMP dependent.

Somatostatin (SRIF) inhibits calcitonin and T3-T4 secretion in thyroid. We have investigated the in vitro effect of SRIF on the basal and TSH induced [3H]thy incorporation, thyroglobulin (tgb) RNA and cAMP level in follicular cells, isolated from normal and adenomatous human thyroids. [3H]thy uptake has been evaluated as TCA-precipitable material in 2, 4, 8, 24 h incubated follicles and 24 h incubated adherent cells. Tgb RNA has been quantified with cytoplasmic dot blot hybridization and cAMP level with RIA method. SRIF reduces basal and TSH-induced [3H]thy in both suspension follicles and epithelial adherent cells. However it does not modify tgb RNA nor cAMP levels in incubated follicles. These data suggest a direct antiproliferative effect of SRIF on human thyroid.

The authors investigated the effects of 17 beta-estradiol (E) and thyroid-stimulating hormone (TSH) on tgb (coding for thyroglobulin), c-myc RNA levels, and [3H]thymidine (thy) incorporation in suspension cultures of normal, adenomatous and carcinomatous human thyroid follicles. The cultured follicles showed decreased tgb RNA and enhanced c-myc RNA levels. In the culture of normal and adenomatous samples E caused a significant increase of [3H]thy incorporation and tgb RNA levels, with no effect on c-myc RNA levels. No effect of E was observed in the carcinomatous thyroid culture. TSH induced a significant increase of [3H]thy incorporation and c-myc expression only in adenoma cultures and a significant increase of tgb RNA levels in both normal and adenomatous samples. TSH had no effect on the carcinoma. The results show that E, like TSH, stimulates in vitro the expression of the tgb gene in differentiated cells, without stimulating the expression of the c-myc proto-oncogene, suggesting a possible action of E on normal thyroid function and perhaps growth, even if not associated with increased c-myc expression.