Evaluation of the timing to reduce the initial dose of antithyroid drugs in patients with Graves' disease.

The present study was undertaken to evaluate whether the normalization of the serum TSH level in a supersensitive assay during the initial treatment with antithyroid drugs (ATD) is a useful indicator for the reduction of the initial dose of ATD in 50 patients with hyperthyroidism due to Graves' disease. The initial dose of ATD was continued until the achievement of the euthyroid state, and was then reduced either before the serum TSH level was in the normal range in 9 of 29 patients treated with methimazole (MMI) (group MMI-1) and 8 of 21 treated with propylthiouracil (PTU) (PTU-1), or after the serum TSH level was in/above the normal range in 20 of 29 treated with MMI (MMI-2) and 13 of 21 treated with PTU (PTU-2). Although there were no significant differences in age, sex, thyroid function, prevalence of autoantibodies, goiter size, duration of the disease or the initial and modified doses of ATD, the mean durations of the administration of the initial dose of ATD in MMI-2 and PTU-2 were significantly longer than those in MMI-1 and PTU-1, respectively. As a result, 4 (44%) in group MMI-1, 20 (100%) in MMI-2, 2 (25%) in PTU-1 and 7 (54%) in PTU-2 developed low free T4 levels, and 1 (11%) in MMI-1, 15 (75%) in MMI-2 and 3 (23%) in PTU-2 developed low free T3 levels. Serum TSH levels increased over the normal range in 3 (33%) in MMI-1, 18 (90%) in MMI-2 and 5 (39%) in PTU-2.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo regulation of hepatic insulin-like growth factor-1 messenger ribonucleic acids with thyroid hormone.

Insulin-like growth factor-1 (IGF-1) is synthesized primarily by the liver in response to growth hormone (GH). Thyroid hormone plays a major role in mediating pituitary GH secretion. In order to clarify the effect of thyroid hormone on IGF-1 gene expression, we measured hepatic IGF-1 mRNA levels in rats with thyroid dysfunction. Female Wistar rats were rendered hypothyroid by surgical thyroidectomy or hyperthyroid by daily injections of thyroxine (12 micrograms/day) for 2 weeks. Northern gel analysis of hepatic poly (A) RNA revealed the multiple sizes of the RNA transcripts ranging from 1.6 to 9.0 kb. After 4 weeks, hepatic IGF-1 mRNA levels were suppressed in hypothyroid rats, to less than 20% of control euthyroid animals. These suppressed mRNA levels were restored to euthyroid levels by thyroid hormone replacement for 2 weeks. Hyperthyroid rats, however, did not contain altered levels of hepatic IGF-1 mRNA as compared to euthyroid rats. The gamma-actin mRNA hybridization signal was not altered in hypothyroid or hyperthyroid rats. These results suggest that thyroid hormone regulates the in vivo expression of hepatic IGF-1 mRNA, probably through the mechanism of GH regulation.

Inhibition of human thyroid peroxidase gene expression by interleukin 1.

We have already demonstrated the inhibitory effect of interleukin 1 on thyroglobulin gene expression. Recent availability of thyroid peroxidase cDNA has allowed us to investigate the regulation of thyroid peroxidase gene. Therefore, the regulation of thyroid peroxidase mRNA by interleukin 1 in cultured human thyrocytes was investigated. Thyrocytes dispersed from thyroid tissues from patients with Graves' disease were incubated with TSH with or without recombinant human interleukin 1. Unstimulated human thyrocytes did not contain any detectable thyroid peroxidase mRNA, however, TSH-stimulated thyrocytes expressed four thyroid peroxidase mRNA transcripts (4.0, 3.2, 2.1 and 1.7 kb, respectively). Both interleukin 1 alpha and beta inhibited TSH-induced thyroid peroxidase mRNA in a dose responsive manner; 10(3) U/1 interleukin 1 caused maximal suppression of TSH-induced thyroid peroxidase mRNA level to nearly basal levels. Interleukin 1 also inhibited cAMP analogue 8-bromo-cyclic AMP induced thyroid peroxidase mRNA level. In contrast the gamma-actin mRNA hybridization signal was not altered in control or treated cells. These results demonstrate that interleukin 1 directly inhibits TSH-induced thyroid peroxidase gene expression and provide further evidence for a paracrine role of interleukin 1 as a local inhibitor of thyroid hormone synthesis.

Interferon-gamma inhibits thyrotropin-induced thyroidal peroxidase gene expression in cultured human thyrocytes.

We have previously demonstrated that interferon-gamma (IFN-gamma) induced HLA-DR antigen and also inhibited thyrotropin (TSH)-induced triiodothyronine (T3) and thyroglobulin (Tg) secretion from cultured human thyrocytes. In order to further clarify the inhibitory effect of IFN-gamma on TSH-stimulated thyroid hormone secretion, we have examined human thyroid peroxidase (TPO) gene expression. Thyrocytes dispersed from Graves' thyroid tissues were incubated with TSH with or without IFN-gamma. Total RNA was extracted, separated and hybridized with 32P-labelled human TPO cDNA. Thyrocytes expressed four TPO mRNA transcripts (4.0, 3.2, 2.1 and 1.7kb, respectively), all of which were stimulated by TSH. IFN-gamma inhibited TSH-stimulated TPO mRNA in a dose dependent manner (0.01-10U/mL). 1 U/mL IFN-gamma caused maximal suppression of TSH-stimulated TPO mRNA levels to basal levels. IFN-gamma also inhibited 8-bromo-cyclic AMP-stimulated TPO mRNA levels. In contrast, the gamma-actin mRNA hybridization signal was not altered in control or treated cells. These results demonstrate that IFN-gamma directly inhibits TSH-stimulated TPO gene expression and provide further support for a role of IFN-gamma as a local modulator of thyroid hormone synthesis.

Interleukin-1 inhibits thyrotrophin-induced human thyroglobulin gene expression.

The regulation of thyroglobulin (Tg) and its specific mRNA by interleukin-1 (IL-1) in cultured human thyrocytes was investigated. Specific binding of 125I-labelled IL-1 on thyrocytes was confirmed by solid-phase binding assay. Thyrocytes dispersed from Graves' thyroid tissues were incubated with TSH with or without recombinant human IL-1. TSH stimulated Tg release from cultured human thyrocytes in a dose- and time-dependent manner. Both IL-1 alpha and beta inhibited TSH-induced Tg release at concentrations ranging from 0.01 to 10 U/ml. The suppressive activities of IL-1 alpha and beta were similar. They did not alter the basal level of Tg release. Unstimulated human thyrocytes did not contain any detectable Tg mRNA, but TSH-stimulated thyrocytes expressed a single species of Tg mRNA (8.5 kb). Both IL-1 alpha and beta inhibited TSH-induced Tg mRNA in a dose-responsive manner. IL-1 (10 U/ml) caused maximal suppression of TSH-induced Tg mRNA to nearly basal levels. In contrast, the gamma-actin mRNA hybridization signal was not altered in control or treated cells. Furthermore, IL-1 stimulated [3H]thymidine uptake into thyrocyte DNA. These results demonstrate that IL-1 directly inhibits TSH-induced Tg gene expression and provide further support for a functional role of IL-1 as a local modulator of thyroid hormone synthesis.

Radioiodine (131I) therapy of Graves' disease: use of the new high resolutional ultrasonic scanner for the determination of the accurate weight of the thyroid gland.

In this series, eighteen patients with Graves' disease were treated with 8000 rads (80 Gy) of radioiodine (131I), using the new high resolutional ultrasonic scanner for the determination of the accurate weight of the thyroid gland. The mean dose of radioiodine administered orally was 4.6 +/- 3.0 mCi (170.2 +/- 110.0 MBq) and 133.7 +/- 44.6 microCi/g (4.95 +/- 1.65 MBq). At one year after treatment, twelve of eighteen patients (66.7%) became euthyroid, five (27.8%) remained hyperthyroid and one (5.6%) became hypothyroid. Analysis of various factors which may be related to the effect of radioiodine therapy revealed that the weight of the thyroid gland in the hyperthyroid and euthyroid groups was significantly different (61.7 +/- 33.5 g vs. 25.1 +/- 9.1 g, p less than 0.05). Furthermore, all patients with larger glands (more than sixty grams) remained hyperthyroid, while the incidence of euthyroidism was as high as 80% in patients with smaller glands (less than forty grams). Although the number of patients studied was small, these results indicate that a larger thyroid gland requires a larger radioiodine dose per gram of tissue than a smaller gland, suggesting that the therapeutic radiation dose should be graded according to the gland size even when the gland size is accurately estimated by ultrasound. Further study is required to determine the appropriate radiation dose graded according to the gland size.

Regulation of thyroid peroxidase and thyroglobulin gene expression by thyrotropin in cultured human thyroid cells.

cDNAs for thyroid peroxidase (TPO) and thyroglobulin (Tg) have been cloned and sequenced. Using such cDNAs, we investigated the regulation of TPO and Tg gene expression by various agents in cultured human thyroid cells. Unstimulated human thyroid cells contained a major RNA species [3.2 kilobases (kb) in length] and several minor RNA species (less than 3.2 kb mRNA) of TPO, but no detectable Tg transcripts. However, TSH-stimulated thyroid cells contained four distinct TPO mRNA species (4.0, 3.2, 2.1, and 1.7 kb) and a single Tg mRNA species (8.5 kb). TSH stimulated the TPO and Tg mRNA levels in a dose- and time-dependent manner. The same results were obtained with 8-bromo-cAMP, a cAMP analog, but not with insulin or insulin-like growth factor I. Furthermore, inductions of these mRNAs by TSH and 8-bromo-cAMP were almost completely blocked by cycloheximide, a protein synthesis inhibitor. These results suggest that human thyroid cells contain four distinct TPO mRNAs and a single species of Tg mRNA, and the levels of all mRNAs are increased by TSH/cAMP stimulation. These increases are blocked by inhibiting protein synthesis, indicating that TSH stimulation of TPO and Tg mRNA levels may be mediated by newly synthesized protein(s).

Glucose stimulation of protooncogene expression and deoxyribonucleic acid synthesis in rat islet cell line.

The rat islet cell line, RIN, established from a transplantable, radiation-induced islet cell tumor represents a unique model to study mechanisms in the control of insulin secretion and biosynthesis. In this study, we have examined the effects of glucose on the protooncogene expressioN and cell growth using a clonal strain of RIN cell, RINr, under serum-free and glucose-free conditions. After 24 h pretreatment, cells were treated with different concentrations of glucose for up to 24 h and then subjected to RNA analysis. Agarose gel electrophoresis of total RNA extracts of RINr cells, followed by hybridization with v-myc DNA yielded a 2.4 kilobase c-myc messenger RNA (mRNA) transcripts. After 24 h pretreatment with serum-free and glucose-free medium, RINr cells expressed a low level of c-myc mRNA transcripts. An increase in c-myc transcripts was detectable within 30 min of D-glucose (200 mg/dl) addition, reaching a maximum of 10-fold within 2 h. Glucose stimulated the steady state of c-myc mRNA transcripts in a dose-responsive manner without any change of gamma-actin mRNA levels after 2 h of treatment. The level of c-myc transcripts then declined as the cells proceeded through G1 to the cycle. [3H]Thymidine uptake into DNA was dramatically increased after 24 h of glucose addition, suggesting that glucose itself stimulates DNA synthesis in RINr cells. These results indicate that glucose-induced proliferation of RINr cells is associated with the stimulation of c-myc gene expression.

Expression and regulation of c-erb-A mRNA from lymphocytes in patients with thyroid dysfunction.

To investigate the expression and the regulation of nuclear triiodothyronine (T3) receptor at the gene level, cellular(c)-erb-A mRNA isolated from lymphocytes in patients with thyroid dysfunction was examined by Northern gel analysis and dot blot hybridization using viral (v)-erb-A cDNA probe. Human lymphocytes contained c-erb-A mRNA (approximately 2.0 kilobase (kb) in length), and c-erb-A mRNA, which was determined by dot blot hybridization, was observed to be increased in hypothyroid patients but unaltered in hyperthyroid patients. The high level of c-erb-A mRNA may contribute in part to the increase in nuclear T3 receptor and these results suggest the presence of up-regulation of nuclear T3 receptor at the gene level in the lymphocytes of hypothyroid patients.

Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase.

The thyroid microsomal antigen (MSA) in autoimmune thyroid disease is a protein of approximately 107 kD. We screened a human thyroid cDNA library constructed in the expression vector lambda gt11 with anti-107-kD monoclonal antibodies. Of five clones obtained, the recombinant beta-galactosidase fusion protein from one clone (PM-5) was confirmed to react with the monoclonal antiserum. The complementary DNA (cDNA) insert from PM-5 (0.8 kb) was used as a probe on Northern blot analysis to estimate the size of the mRNA coding for the MSA. The 2.9-kb messenger RNA (mRNA) species observed was the same size as that coding for human thyroid peroxidase (TPO). The probe did not bind to human liver mRNA, indicating the thyroid-specific nature of the PM-5-related mRNA. The nucleotide sequence of PM-5 (842 bp) was determined and consisted of a single open reading frame. Comparison of the nucleotide sequence of PM-5 with that presently available for pig TPO indicates 84% homology. In conclusion, a cDNA clone representing part of the microsomal antigen has been isolated. Sequence homology with porcine TPO, as well as identity in the size of the mRNA species for both the microsomal antigen and TPO, indicate that the microsomal antigen is, at least in part, TPO.

Initiation of normal thyroid cells in primary culture associated with enhanced c-myc messenger ribonucleic acid levels.

We studied expression of the c-myc and c-ras protooncogenes during the initiation of pig thyroid cells in primary cell culture. This period is associated with major changes in differentiated thyroid cell function. After 1, 2, and 3 days of culture, polyadenylated mRNA was prepared from 30-50 replicate dishes of cells. mRNA was also prepared from a portion of the intact tissue used to prepare the dispersed follicles. Expression of c-myc and c-ras mRNA was determined by Northern blot analysis using v-myc and v-ras probes labeled by nick translation. As a nononcogene control, actin mRNA was determined using a beta-actin probe. In intact thyroid tissue before follicle dispersion, c-myc (2.6 kilobases) and c-ras (1.1 kilobases) mRNA were detectable, though at relatively low levels. c-myc mRNA expression increased in cultured cells, to 430%, 670%, and 330% of tissue levels on the first, second, and third days of culture, respectively. In contrast to c-myc, c-ras oncogene mRNA expression was not significantly altered during the 3-day culture period. beta-Action mRNA expression, like c-myc, increased to 560%, 810%, and 460% of tissue levels on the first, second, and third days of culture, respectively. Southern blot analysis of thyroid tissue and cultured cell DNA indicated that gene amplification did not account for the increase in c-myc mRNA levels in cultured cells. In conclusion, c-myc, but not c-ras, oncogene expression is enhanced during the transition of thyroid follicular cells into monolayer culture. beta-Actin mRNA expression is also enhanced during the initiation of primary thyroid cell culture. These data are consistent with a role for c-myc expression in the regulation of normal thyroid cell differentiated function.

Molecular cloning and partial characterization of a new autoimmune thyroid disease-related antigen.

To clone and characterize antigens to autoantibodies in Hashimoto's thyroiditis we constructed a cDNA library in the expression vector lambda gt11 using mRNA prepared from Grave's thyroid tissue. This library was screened using serum from a patient with Hashimoto's thyroiditis which had an antimicrosomal antibody titer greater than 1:10(6). Five positive recombinants were identified and cloned. Of these, 3 reacted with 7 of 17 normal serum samples. The 2 other clones (IL-28 and IL-33) reacted with none of the 17 normal serum samples. IL-28 reacted with 4 of 15 and IL-33 with 2 of 15 Hashimoto's thyroiditis serum samples (antimicrosomal antibody titers, greater than 1:6400). The specificity of the interaction between the Hashimoto's thyroiditis samples and the fusion protein was demonstrated by Western blot analysis. In addition, neither 10(-6) M human thyroglobulin nor 100 mU/ml bovine TSH inhibited binding of the serum samples to these 2 clones. Lysate from clones IL-28 and IL-33 did not reduce the antimicrosomal antibody titer in a hemagglutination assay. Absorption of Hashimoto's thyroiditis serum with purified thyroid microsomes reduced the serum antimicrosomal antibody titer, but not binding to these 2 clones. The cDNA inserts of clones IL-28 and IL-33 were approximately 0.6 and 0.4 kilobases (kb), respectively. The 0.6-kb IL-28 insert was used to probe human thyroid and human liver poly(A)+ mRNA. A single band of 3.3 kb was evident only with the thyroid mRNA. The IL-28 insert was subcloned into M13 and sequenced in both directions by the dideoxy technique and found to be 572 basepairs in length. When tested against the GenBank and Dayhoff gene banks, no significant homology with any known sequence was determined. In summary, a cDNA fragment of a previously unrecognized gene coding for an autoimmune thyroid disease-related antigen has been cloned and partly characterized; and the protein produced by this clone is not thyroglobulin, the thyroid microsomal antigen, or the TSH-binding site of the TSH receptor. We have, therefore, identified a new autoimmune thyroid disease-related antigen, the pathogenetic significance of which remains to be determined.

Molecular cloning of antigens to thyroid autoantibodies using the expression vector lambda gt11.

The molecular cloning of certain antigens to thyroid autoantibodies present in patients with autoimmune thyroid disease would be of great value in further understanding the pathogenesis of these diseases, and in devising new approaches for their treatment. The ideal method for molecular cloning is to first obtain a partial amino acid sequence of a purified protein and to construct an oligonucleotide probe for screening an appropriate cDNA library. Unfortunately in the case if thyroid autoimmunity, important antigens such as the TSH receptor and the microsomal antigen have not been purified. An alternate approach devised by Young & Davis (1983) is to construct a cDNA library in a vector that expresses the encoded proteins. The library can then be screened with an antibody as a probe. We have constructed cDNA libraries in gt11 using mRNA purified from human, pig and rat thyroid cells. Our experiences in constructing and screening these libraries will be described. The advantages of this system are 1) the protein does not have to be purified, 2) previously unknown antigens may be identified. The disadvantages are 1) lack of specificity with antibody selection, 2) because the cDNA is inserted in the beta-galactosidase gene in the vector the antigen is expressed as a fusion protein. This may disturb the tertiary structure of the antigen and alter its antigenicity, 3) cDNA inserts frequently only contain part of the antigen molecule, and may therefore lack important epitopes; polyclonal antibody may therefore be preferable to monoclonal, 4) only 1 in 6 cDNA inserts will be in the correct reading frame for antigen expression, 5) the expressed protein is not glycosylated.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin and cyclic AMP regulation of ras proto-oncogene expression in cultured thyroid cells.

We examined the effect of thyrotropin (TSH) on intracellular levels of c-ras mRNA in a line of differentiated rat thyroid cells obtained from normal Fischer rat thyroids. These cells are totally dependent on TSH for growth. TSH stimulation of quiescent cells increased c-ras mRNA content, with a maximal response (730% of basal) after 6 h, and a decline towards basal levels after 24 h. Dibutyryl cAMP and forskolin mimicked this stimulatory effect of TSH on c-ras, but did not enhance beta-actin mRNA content. This study demonstrates hormonal and cyclic nucleotide control of c-ras expression in a well-differentiated, non-tumorogenic mammalian cell.

TSH and cAMP enhance expression of the myc proto-oncogene in cultured thyroid cells.

The effect of TSH stimulation on cellular (c)-myc mRNA content in cultured thyroid cells was examined by Northern blot analysis. Polyadenylated mRNA was prepared from quiescent FRTL-5 rat thyroid cells and from cells stimulated by TSH for 8 h. A major 2.1 kilobase (kb) transcript was enhanced 5.5-fold by TSH. (Bu)2cAMP and forskolin each enhanced c-myc expression 2.7-fold. These data indicate that TSH, via cAMP as a second messenger, can regulate expression of a cellular oncogene in a non-neoplastic, specific target cell.

Studies on the mechanism of desensitization of the cyclic AMP response to TSH stimulation in a cloned rat thyroid cell line.

We examined aspects of the mechanism of desensitization of adenylate cyclase activation by TSH in a cloned line of rat thyroid cells (FRTL). Increasing FRTL intracellular cAMP concentrations by preincubation for 6 h in either 1 mM dBcAMP or 100 microM forskolin did not induce TSH desensitization. Forskolin stimulation was unimpaired in TSH-desensitized cells, indicating 'uncoupling' of the adenylate cyclase catalytic unit from the TSH receptor. Stimulation by the Ni inhibitory pathway of the adenylate cyclase by epinephrine (10(-6) M-10(-4) M in the presence of 10(-4) M propranolol) was unaltered in cells previously desensitized to TSH. That is, Ni-mediated inhibition of adenylate cyclase was additive to TSH desensitization. Pre-exposure of FRTL cells for 18 h to 50 ng/ml pertussis toxin did not prevent the induction of TSH desensitization. TSH desensitization was prevented by cycloheximide or actinomycin D added during the last 3-4 h of a 6 h period of TSH stimulation. The rates of turnover of the putative desensitization protein and its mRNA therefore appear to be similar.