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E C Ridgway

Publications and source records attributed to E C Ridgway.

At least 19 recordsLinked to original sources
PubMed2001

Signaling pathways responsible for fetal gene induction in the failing human heart: evidence for altered thyroid hormone receptor gene expression.

BACKGROUND: We have previously demonstrated that changes in myosin heavy chain (MHC) isoforms that occur in failing human hearts resemble the pattern produced in rodent myocardium in response to hypothyroidism. Because thyroid hormone status is usually within normal limits in these patients, we hypothesized that failing/hypertrophied human myocardium might have a defect in thyroid hormone signaling due to alterations in expression of thyroid hormone receptors (TRs). METHODS AND RESULTS: To examine this hypothesis, we used RNase protection assay to measure mRNA levels of TRs in failing left ventricles that exhibited a fetal pattern of gene expression, ie, decreased expression of alpha-MHC with increased beta-MHC expression compared with left ventricles from age-matched controls. We detected expression of TR-alpha(1), -alpha(2), and -beta(1) isoforms in human left ventricles. In failing left ventricles, TR-alpha(1) was downregulated, whereas TR-alpha(2), a splice variant that does not bind thyroid hormone but inhibits responses to liganded TRs, was increased. Expression levels of TR-beta(1) did not differ significantly between the 2 groups. According to linear regression analysis, expression levels of TR-alpha(1) and -alpha(2) were positively and negatively correlated with those of alpha-MHC, respectively. CONCLUSIONS: We conclude that decreases in TR-alpha(1) and increases in TR-alpha(2) may lead to local attenuation of thyroid hormone signaling in the failing human heart and that the resulting tissue-specific hypothyroidism is a candidate for the molecular mechanism that induces fetal gene expression in the failing human ventricle.

Adult↗
PubMed2000

The Colorado thyroid disease prevalence study.

CONTEXT: The prevalence of abnormal thyroid function in the United States and the significance of thyroid dysfunction remain controversial. Systemic effects of abnormal thyroid function have not been fully delineated, particularly in cases of mild thyroid failure. Also, the relationship between traditional hypothyroid symptoms and biochemical thyroid function is unclear. OBJECTIVE: To determine the prevalence of abnormal thyroid function and the relationship between (1) abnormal thyroid function and lipid levels and (2) abnormal thyroid function and symptoms using modern and sensitive thyroid tests. DESIGN: Cross-sectional study. PARTICIPANTS: Participants in a statewide health fair in Colorado, 1995 (N = 25 862). MAIN OUTCOME MEASURES: Serum thyrotropin (thyroid-stimulating hormone [TSH]) and total thyroxine (T4) concentrations, serum lipid levels, and responses to a hypothyroid symptoms questionnaire. RESULTS: The prevalence of elevated TSH levels (normal range, 0.3-5.1 mIU/L) in this population was 9.5%, and the prevalence of decreased TSH levels was 2.2%. Forty percent of patients taking thyroid medications had abnormal TSH levels. Lipid levels increased in a graded fashion as thyroid function declined. Also, the mean total cholesterol and low-density lipoprotein cholesterol levels of subjects with TSH values between 5.1 and 10 mIU/L were significantly greater than the corresponding mean lipid levels in euthyroid subjects. Symptoms were reported more often in hypothyroid vs euthyroid individuals, but individual symptom sensitivities were low. CONCLUSIONS: The prevalence of abnormal biochemical thyroid function reported here is substantial and confirms previous reports in smaller populations. Among patients taking thyroid medication, only 60% were within the normal range of TSH. Modest elevations of TSH corresponded to changes in lipid levels that may affect cardiovascular health. Individual symptoms were not very sensitive, but patients who report multiple thyroid symptoms warrant serum thyroid testing. These results confirm that thyroid dysfunction is common, may often go undetected, and may be associated with adverse health outcomes that can be avoided by serum TSH measurement.

Adult↗
PubMed2000

The effect of thyroid hormone and a long-acting somatostatin analogue on TtT-97 murine thyrotropic tumors.

Thyroid hormone inhibits thyrotropin (TSH) production and thyrotrope growth. Somatostatin has been implicated as a synergistic factor in the inhibition of thyrotrope function. We have previously shown that pharmacological doses of thyroid hormone (levothyroxine [LT4]) inhibit growth of murine TtT-97 thyrotropic tumors in association with upregulation of somatostatin receptor type 5 (sst5) mRNA and somatostatin receptor binding. In the current study, we examined the effect of physiological thyroid hormone replacement alone or in combination with the long-acting somatostatin analogue, Sandostatin LAR, on thyrotropic tumor growth, thyrotropin growth factor-beta (TSH-beta), and sst5 mRNA expression, as well as somatostatin receptor binding sites. Physiological LT4 replacement therapy resulted in tumor shrinkage in association with increased sst5 mRNA levels, reduced TSH-beta mRNA levels and enhanced somatostatin receptor binding. Sandostatin LAR alone had no effect on any parameter measured. However, Sandostatin LAR combined with LT4 synergistically inhibited TSH-beta mRNA production and reduced final tumor weights to a greater degree. In this paradigm, Sandostatin LAR required a euthyroid status to alter thyrotrope parameters. These data suggest an important interaction between the somatostatinergic system and thyroid hormone in the regulation of thyrotrope cell structure and function.

Animals↗
PubMed1999

An upstream regulator of the glycoprotein hormone alpha-subunit gene mediates pituitary cell type activation and repression by different mechanisms.

Targeting of alpha-subunit gene expression within the pituitary is influenced by an upstream regulatory region that directs high level expression to thyrotropes and gonadotropes of transgenic mice. The same region also enhanced the activity of the proximal promoter in transfections of pituitary-derived alpha-TSH and alpha-T3 cells. We have localized the activating sequences to a 125-bp region that contains consensus sites for factors that also play a role in proximal promoter activity. Proteins present in alpha-TSH and alpha-T3 cells as well as those from GH3 somatotrope-derived cells interact with this region. The upstream area inhibited proximal alpha-promoter activity by 80% when transfected into GH3 cells. Repression in GH3 cells was mediated through a different mechanism than enhancement, as supported by the following evidence. Reversing the orientation of the area resulted in a loss of proximal promoter activation in alpha-TSH and alpha-T3 cells but did not relieve repression in GH3 cells. Mutation of proximal sites shown to be important for activation had no effect on repression. Finally, bidirectional deletional analysis revealed that multiple elements are involved in activation and repression and, together with the DNA binding studies, suggests that these processes may be mediated through closely juxtaposed or even overlapping elements, thus perhaps defining a new class of bifunctional gene regulatory sequence.

Animals↗
PubMed1999

Reverse transcription polymerase chain reaction analysis of pituitary hormone, Pit-1 and steroidogenic factor-1 messenger RNA expression in pituitary tumors.

Pit-1 is a transcription factor that appears early in embryonic pituitary gland formation and is necessary for the development of somatotropes, lactotropes and thyrotropes. Steroidogenic factor-1 (SF-1) is another early appearing transcription factor that is involved in the development of gonadotropes. In this study we have compared RT-PCR analysis of hormone mRNA with traditional IHC for classification of 27 pituitary tumors and have evaluated the correlation of Pit-1 and SF-1 mRNA with hormone mRNA. RT-PCR detected concordant hormone mRNA in 100% of GH IHC positive, 100% of PRL IHC positive, 33% of TSH IHC positive, and 93% of gonadotropin IHC positive tumors. IHC, however, was concordant in only 71% of GH mRNA positive, 78% of PRL mRNA positive, 17% of TSH beta mRNA positive, and 76% of FSH beta mRNA positive tumors. Pit-1 mRNA was positive in 87% of tumors in which mRNA for GH, PRL or TSH beta was detected and in only 17% of GH, PRL and TSH beta mRNA negative tumors. SF-1 mRNA was positive in 94% of tumors in which mRNA for FSH beta was present and in no FSH beta mRNA negative tumors. We conclude that RT-PCR analysis of hormone mRNA may be more sensitive than traditional hormone IHC for classification of pituitary tumors. Furthermore, tumor Pit-1 mRNA positively correlates with GH, PRL and TSH beta mRNA while tumor SF-1 mRNA correlates well with FSH beta mRNA. Combined analysis of hormone and transcription factor mRNA in pituitary tumor tissue may therefore be a more meaningful approach to pituitary tumor characterization.

DNA Primers↗
PubMed1999

Cloning of the mouse somatostatin receptor subtype 5 gene: promoter structure and function.

Somatostatin is a peptide hormone whose actions are mediated by five somatostatin receptor subtypes (sstl-5). In the pituitary, somatostatin inhibits TSH release from thyrotropes and GH release from somatotropes. We have shown that sst5 transcripts and protein are induced by thyroid hormone in TtT-97 thyrotropic tumors. To map sequences responsible for promoter activity in pituitary cells, we cloned the mouse sst5 coding region of 362 amino acids and 12 kb of upstream DNA. Initial transfection studies in TtT-97 or GH3 cells mapped high levels of basal promoter activity to a 5.6-kb fragment upstream of the translational start, whereas shorter genomic fragments had low activity. To identify the transcriptional start site we used 5' RACE with TtT-97 poly A+ RNA and a sst5 antisense coding region primer. Sequence comparison between the complementary DNA and the gene revealed that the mouse sst5 gene contains 3 exons and 2 introns. The entire coding region was contained in exon 3. Two differently sized RACE products demonstrated alternate exon splicing of two untranslated exons in TtT-97 cells. A promoter fragment from -290/+48 linked to a luciferase reporter demonstrated 600- and 900-fold higher activity over a promoterless control in GH3 mammosomatotropes and TtT-97 thyrotropes, respectively, whereas a larger fragment extending to -6400 exhibited no additional promoter activity. Cloning of the sst5 gene will facilitate the mapping of basal and regulated responses at the transcriptional level.

Amino Acid Sequence↗
PubMed1999

A comparison of recombinant human thyrotropin and thyroid hormone withdrawal for the detection of thyroid remnant or cancer.

Recombinant human TSH has been developed to facilitate monitoring for thyroid carcinoma recurrence or persistence without the attendant morbidity of hypothyroidism seen after thyroid hormone withdrawal. The objectives of this study were to compare the effect of administered recombinant human TSH with thyroid hormone withdrawal on the results of radioiodine whole body scanning (WBS) and serum thyroglobulin (Tg) levels. Two hundred and twenty-nine adult patients with differentiated thyroid cancer requiring radioiodine WBS were studied. Radioiodine WBS and serum Tg measurements were performed after administration of recombinant human TSH and again after thyroid hormone withdrawal in each patient. Radioiodine whole body scans were concordant between the recombinant TSH-stimulated and thyroid hormone withdrawal phases in 195 of 220 (89%) patients. Of the discordant scans, 8 (4%) had superior scans after recombinant human TSH administration, and 17 (8%) had superior scans after thyroid hormone withdrawal (P = 0.108). Based on a serum Tg level of 2 ng/mL or more, thyroid tissue or cancer was detected during thyroid hormone therapy in 22%, after recombinant human TSH stimulation in 52%, and after thyroid hormone withdrawal in 56% of patients with disease or tissue limited to the thyroid bed and in 80%, 100%, and 100% of patients, respectively, with metastatic disease. A combination of radioiodine WBS and serum Tg after recombinant human TSH stimulation detected thyroid tissue or cancer in 93% of patients with disease or tissue limited to the thyroid bed and 100% of patients with metastatic disease. In conclusion, recombinant human TSH administration is a safe and effective means of stimulating radioiodine uptake and serum Tg levels in patients undergoing evaluation for thyroid cancer persistence and recurrence.

Adult↗
PubMed1998

Activation of the glycoprotein hormone alpha-subunit gene promoter in thyrotropes.

Pituitary expression of the glycoprotein hormone alpha-subunit gene localizes to thyrotropes and gonadotropes. Factors that activate the alpha-subunit promoter exclusively in thyrotropes have not been described. In the current studies, alpha-subunit promoter activity was compared in alphaTSH thyrotropic and alphaT3 gonadotropic cells. Deletional analysis revealed a more important contribution of the -453/-381 region in thyrotropic cells and the -341/-297 region in gonadotropic cells, while other deletions had similar effects. Mutational analysis revealed some regions that were functionally similar, while others were active only in thyrotropic cells, such as the regions - 434/ - 421, - 398/ - 385, - 376/- 364 and - 363/- 351, that decreased activity by approximately 2-fold each. Combined mutation of the regions - 434/ - 421, - 398/ - 385 and - 376/ - 364 decreased activity by 5-fold in thyrotropic cells and not at all in gonadotropic cells. Cotransfection with Ptx1 in CV-1 cells resulted in a strong stimulation of alpha-subunit promoter activity, that was significantly diminished with mutation of the - 398/ - 385 region that disrupts the Ptx1 binding site, suggesting that this factor may play a role in thyrotrope-specific activation. This analysis provides basic information important to identify common and unique factors contributing to the cell-specific expression of the alpha-subunit gene.

Animals↗
PubMed1998

Functional interactions of an upstream enhancer of the mouse glycoprotein hormone alpha-subunit gene with proximal promoter sequences.

Transcription of the glycoprotein hormone alpha-subunit gene in the pituitary is governed by different promoter elements in thyrotropes and gonadotropes. We recently identified an upstream enhancer that directs a high level of cell type specific expression in transgenic mice and stimulates proximal promoter activity in cultured alphaTSH and alphaT3 cells. To assess the contribution of promoter sequences that functionally interact with the enhancer, we mutated two proximal elements shown to be important in both thyrotrope and gonadotrope cells. Disruption of the pituitary glycoprotein hormone basal element (PGBE), which binds a LIM homeodomain protein, resulted in a decrease in basal promoter activity in both alphaTSH and alphaT3 cells. Enhancer function was completely abolished by the PGBE site mutation in alphaT3 gonadotropes, whereas some stimulatory activity remained in alphaTSH thyrotropes. Mutation of the gonadotrope specific element (GSE), which binds SF1 and is important for basal activity in gonadotropes and TRH response in thyrotropes, resulted in declines in basal and enhanced promoter activity only in alphaT3 cells and not in alphaTSH cells. Despite this decrease in enhanced activity, the GSE mutated promoter still retained some enhancer stimulated activity, suggesting that the PGBE site still functionally interacts in the absence of an intact GSE. This mutation had no effect in alphaTSH cells. These data suggest that although the enhancer works in both cell types it exhibits cell type specific functional characteristics.

Animals↗
PubMed1998

Central hyperthyroidism.

Central hyperthyroidism is a rare condition in which thyrotoxicosis results from primary overproduction of TSH by the pituitary gland with subsequent thyroid enlargement and hyperfunction. The two known causes of central hyperthyroidism are TSH-producing pituitary tumors (TSHomas) and the syndrome of PRTH. Both of these entities are characterized by clinical thyrotoxicosis, diffuse goiters, elevated circulating levels of free T4 and T3, and a nonsuppressed serum TSH. It is critical to distinguish central hyperthyroidism from the much more common types of primary hyperthyroidism, all of which have undetectable TSH values. TSHomas and PRTH can usually be differentiated from one another by measuring the serum alpha-subunit and the TSH response to intravenous TRH or exogenous thyroid hormone, and by pituitary imaging studies. TSHomas are usually benign adenomas arising from the monoclonal expansion of neoplastic thyrotropes. Causative oncogenes have not yet been convincingly identified. PRTH is a nonneoplastic disorder caused by inherited mutations in the gene for the thyroid hormone receptor beta; it is a poorly understood variant of GRTH. For unclear reasons, in PRTH, the pituitary gland is resistant to the feedback inhibitory effects of circulating thyroid hormones while peripheral tissues respond normally, causing patients to experience the toxic peripheral effects of thyroid hormone excess. TSHomas are best treated by transphenoidal surgical removal. Radiotherapy is indicated for inoperable or incompletely resected tumors. Octreotide administration is a useful adjunct for preoperatively reducing tumor size and for the medical management of surgical treatment failures. PRTH is ideally treated by chronically suppressing TSH secretion with medications such as D-thyroxine, TRIAC, octreotide, or bromocriptine. If such therapy is ineffective or unavailable, thyroid ablation with radioiodine or surgery may be employed with subsequent close monitoring of both thyroid hormone status and pituitary gland size.

Adenoma↗
PubMed1998

Reassessment of the role of radiation therapy in the treatment of endocrine-inactive pituitary macroadenomas.

OBJECTIVE: This prospective clinical trial was undertaken to assess the rate of tumor recurrence in patients with endocrine-inactive pituitary macroadenomas who underwent gross total surgical resection of their tumors and did not receive adjuvant radiotherapy. METHODS: Between December 1987 and July 1994, 45 patients with endocrine-inactive pituitary macroadenomas underwent transsphenoidal surgery. In 38 (84%) of these patients, gross total surgical resection was achieved and was confirmed by postoperative magnetic resonance imaging (n = 37) or computed tomography (n = 1). After receiving counseling from the neurosurgeon concerning the risks and benefits of radiation therapy, 32 of the 38 patients elected not to receive adjuvant radiotherapy. Patients were followed through March 1998 with radiographic imaging obtained every 6 months for the first 2 years, annually for postoperative Years 3 and 4, and then every 2 to 3 years thereafter. The study end point was defined as radiographic tumor recurrence or patient death. RESULTS: The mean follow-up duration for the study group was 5.5 years. During that time, 2 of 32 (6%) patients developed recurrence, at 18 and 24 months, respectively, after initial surgery. Both were successfully treated using radiation therapy, with one requiring additional surgery. Three additional patients died as a result of unrelated causes 9, 12, and 49 months, respectively, after initial surgery. Immunocytochemical analysis revealed 66% of the tumors to be weak gonadotroph cell adenomas, 22% to be null cell adenomas, 9% to be silent prolactinomas, and 3% to be silent corticotroph cell adenomas. CONCLUSION: This study demonstrates a 6% 5-year recurrence rate in patients with endocrine-inactive pituitary macroadenomas treated using gross total surgical resection alone. Reserving radiation therapy for the infrequent patient with recurrence and sparing the majority of patients the associated risks inherent in its use seems reasonable.

Adenoma↗
PubMed1998

The thyrotropin beta-subunit gene is repressed by thyroid hormone in a novel thyrotrope cell line, mouse T alphaT1 cells.

TSH is expressed in two populations of thyrotropes in the pituitary: one in the pars distalis and a second in the pars tuberalis. Pars distalis thyrotropes exhibit classical endocrine inhibition of TSH by thyroid hormone, whereas pars tuberalis thyrotropes do not. The majority of our understanding of TSH subunit gene regulation has come from studies conducted in dispersed pituitary, dispersed thyrotropic tumors, or the GH3 somatolactotrope cell line. However, the dispersed pituitary model is limited because of its inherent heterogeneity, thyrotropic tumors are difficult to grow and maintain, and the GH3 cells lack endogenous TSH expression. The recent derivation of a clonal thyrotrope cell line, T alphaT1, that expresses thyrotrope-specific markers, overcomes these limitations. However, because it was not possible to distinguish whether the tumor from which the T alphaT1 cells are derived originated in the pars distalis or the pars tuberalis, it was necessary to define their cellular origin and thereby establish their status as representative thyrotrope cells for future molecular studies. In this study, we demonstrate that the T alphaT1 cells express thyroid hormone receptors (beta1 and beta2) and their heterodimeric partner, retinoid X receptor-gamma. Treatment with T3 causes a dose- and time-dependent decrease in the expression of the TSH beta-subunit messenger RNA. In contrast to previous reports in rat pituitary cultures, T3 does not alter TSH beta-subunit messenger RNA stability in the T alphaT1 cells. Based on these data and the presence of thyrotrope-specific isoforms of the transcription factor Pit-1, we conclude that the T alphaT1 cells represent differentiated thyrotropes of the pars distalis and will be a useful model system for future analysis of the cis- and trans-acting factors necessary for thyrotrope-specific and thyroid hormone-regulated TSH gene expression.

Animals↗
PubMed1998

Cell-specific expression of the mouse glycoprotein hormone alpha-subunit gene requires multiple interacting DNA elements in transgenic mice and cultured cells.

The glycoprotein hormone alpha-subunit gene is expressed and differentially regulated in pituitary gonadotropes and thyrotropes. Previous gene expression studies suggested that cell specificity may be regulated by distinct DNA elements. We have identified an enhancer region between -4.6 and -3.7 kb that is critical for high level expression in both gonadotrope and thyrotrope cells of transgenic mice. Fusion of the enhancer to -341/+43 mouse alpha-subunit promoter results in appropriate pituitary cell specificity and transgene expression levels that are similar to levels observed with the intact -4.6 kb/+43 construct. Deletion of sequences between -341 and -297 resuited in a loss of high level expression and cell specificity, exhibited by ectopic transgene activation in GH-, ACTH-, and PRL-producing pituitary cells as well as in other peripheral tissues. Consistent with these results, transient cell transfection studies demonstrated that the enhancer stimulated activity of a -341/+43 alpha-promoter in both alphaTSH and alphaT3 cells, but it did not enhance alpha-promoter activity significantly in CV-1 cells. Removal of sequences between -341 and -297 allowed the enhancer to function in heterologous cells. Loss of high level expression and cell specificity may be due to loss of sequences required for binding of the LIM homeoproteins or the alpha-basal element 1. These data demonstrate that the enhancer requires participation by both proximal and distal sequences for high level expression and suggests that sequences from -341 to -297 are critical for restricting expression to the anterior pituitary.

Animals↗
PubMed1997

Pit-1 and GATA-2 interact and functionally cooperate to activate the thyrotropin beta-subunit promoter.

The molecular determinants governing cell-specific expression of the thyrotropin (TSH) beta-subunit gene in pituitary thyrotropes are not well understood. The P1 region of the mouse TSHbeta promoter (-133 to -88) region interacts with Pit-1 and an additional 50-kDa factor at an adjacent site that resembles a consensus GATA binding site. Northern and Western blot assays demonstrated the presence of GATA-2 transcripts and protein in TtT-97 thyrotropic tumors. In electrophoretic mobility shift assays, a comigrating complex was observed with both TtT-97 nuclear extracts and GATA-2 expressed in COS cells. The complex demonstrated binding specificity to the P1 region DNA probe and could be disrupted by a GATA-2 antibody. When both Pit-1 and GATA-2 were combined, a slower migrating complex, indicative of a ternary protein-DNA interaction was observed. Cotransfection of both Pit-1 and GATA-2 into CV-1 cells synergistically stimulated mouse TSHbeta promoter activity 8.5-fold, while each factor alone had a minimal effect. Mutations that abrogated this functional stimulatory effect mapped to the P1 region. Finally, we show that GATA-2 directly interacts with Pit-1 in solution. In summary, these data demonstrate functional synergy and physical interaction between homeobox and zinc finger factors and provide insights into the transcriptional mechanisms of thyrotrope-specific gene expression.

Animals↗
PubMed1997

Do traditional symptoms of hypothyroidism correlate with biochemical disease?

OBJECTIVE: Hypothyroidism often remains undetected because of the difficulty associating symptoms with disease. To determine the relation between symptoms and biochemical disease, we assessed symptoms and serum thyroid function tests, concurrently, for patients with and without hypothyroidism. DESIGN: Cross-sectional study. SETTING/PATIENTS: Seventy-six newly diagnosed case patients with overt hypothyroidism and 147 matched control patients identified through outpatient laboratories in Michigan and Colorado. MEASUREMENTS AND MAIN RESULTS: Patient symptoms were assessed by questionnaire. Case patients reported a higher proportion of hypothyroid symptoms than did control patients (30.2% vs 16.5%, p < .0001). Univariate analysis identified three significant predictors of an elevated level of thyroid-stimulating hormone (TSH) (p < .05), and 13 symptoms which, when they had changed in the past year, were reported more often by case patients with hypothyroidism than by control patients (p < .005). Individuals reporting changes in 7 or more symptoms were significantly more likely to have hypothyroidism (likelihood ratio [LR] = 8.7, 95% confidence interval [CI] 3.8, 20.2); those reporting changes in 2 or fewer symptoms were less likely to have hypothyroidism (LR = 0.5, 95% CI 0.4, 0.7). CONCLUSIONS: In this sample, the number of hypothyroid symptoms reported was directly related to the level of TSH. The association was stronger when more symptoms were reported. Symptoms that had changed in the past year were more powerful than symptoms reported present at the time of testing. This suggests that traditional symptoms are valuable when deciding which patients to test for hypothyroidism.

Adult↗
PubMed1997

Reconstitution of triiodothyronine inhibition in non-triiodothyronine-responsive thyrotropic tumor cells using transfected thyroid hormone receptor isoforms.

The triiodothyronine (T3) inhibitory effect on the thyrotropin (TSH)beta- and alpha-subunit genes is believed to be mediated by binding of T3 to specific nuclear receptors that are present in various isoforms. alphaTSH cells, which are derived from a pure alpha-subunit secreting thyrotropic tumor, contain the same nuclear factors that are important for alpha-subunit gene expression in TSH-expressing T3-responsive thyrotropic cells (TtT97). However, as in the parent tumor, alpha-subunit expression in alphaTSH cells was not inhibited by T3, despite the presence of high-affinity nuclear T3 receptors (TRs) with a similar number of sites per cell as in TtT97. When transcripts coding for the different TR isoforms from the MGH101A tumor were analyzed by Northern blot, TR alpha1 was present, as well as the non-T3-binding variant alpha2, but transcripts encoding the opposite strand Rev-ErbAa were not detectable and neither TR beta1 nor TR beta2 mRNAs were detectable, whereas all these transcripts were detectable in TtT97 tumors. Similar findings were observed in alphaTSH cells, where TR beta1 transcripts were barely detectable in Northern blots and TR beta2 transcripts were detectable only by RT-PCR. The TR beta gene locus is present and unrearranged in the tumor genome. In transient transfection studies conducted in alphaTSH cells overexpression of either TR beta1, TR beta2, or TR alpha1 reconstituted T3-inhibition of the alpha-subunit promoter down to 40% to 50% of control. We conclude that the relative lack of TR beta gene expression correlates with unresponsiveness to T3. The alphaTSH cell line represents a unique model in which to dissect the mechanism of T3 inhibition.

Blotting, Northern↗