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Biomedical subjects

G A Brent

Publications and source records attributed to G A Brent.

At least 19 recordsLinked to original sources
PubMed2001

Ectopic expression of the thyroperoxidase gene augments radioiodide uptake and retention mediated by the sodium iodide symporter in non-small cell lung cancer.

Radioiodide is an effective therapy for thyroid cancer. This treatment modality exploits the thyroid-specific expression of the sodium iodide symporter (NIS) gene, which allows rapid internalization of iodide into thyroid cells. To test whether a similar treatment strategy could be exploited in nonthyroid malignancies, we transfected non-small cell lung cancer (NSCLC) cell lines with the NIS gene. Although the expression of NIS allowed significant radioiodide uptake in the transfected NSCLC cell lines, rapid radioiodide efflux limited tumor cell killing. Because thyroperoxidase (TPO) catalyzes iodination of proteins and subsequently causes iodide retention within thyroid cells, we hypothesized that coexpression of both NIS and TPO genes would overcome this deficiency. Our results show that transfection of NSCLC cells with both human NIS and TPO genes resulted in an increase in radioiodide uptake and retention and enhanced tumor cell apoptosis. These findings suggest that single gene therapy with only the NIS gene may have limited efficacy because of rapid efflux of radioiodide. In contrast, the combination of NIS and TPO gene transfer, with resulting TPO-mediated organification and intracellular retention of radioiodide, may lead to more effective tumor cell death. Thus, TPO could be used as a therapeutic strategy to enhance the NIS-based radioiodide concentrator gene therapy for locally advanced lung cancer.

Apoptosis↗
PubMed2001

Regulation of prohormone convertase 1 (PC1) by thyroid hormone.

The prohormone convertases (PCs) PC1 and PC2 are key enzymes capable of processing a variety of prohormones to their bioactive forms. In this study, we demonstrated that 6-n-propyl-2-thiouracil (PTU)-induced hypothyroidism stimulated, whereas triido-L-thyronine (T(3))-induced hyperthyroidism suppressed, PC1 mRNA levels in the rat anterior pituitary. Using 5' deletions of the human PC1 (hPC1) promoter transiently transfected into GH3 (a somatotroph cell line) cells, we found that T(3) negatively regulated hPC1 promoter activity and that this regulation required the region from -82 to +19 bp relative to the transcription start site. Electrophoretic mobility shift assays (EMSAs) using purified thyroid hormone receptor-alpha1 (TR alpha 1) and retinoid X receptor-beta (RXRbeta) proteins and GH3 nuclear extracts demonstrated that the region from -10 to +19 bp of the hPC1 promoter bound TR alpha 1 as both a monomer and a homodimer and bound TR alpha 1/RXR beta as a heterodimer and multimer. EMSAs with oligonucleotides containing point mutations of the putative negative thyroid response elements (TREs) exhibited diminished homodimer and loss of multimer binding. We conclude that there are multiple novel TRE-like sequences in the hPC1 promoter located from -10 to +19 bp.

Alitretinoin↗
PubMed2001

Thyroid hormone--sympathetic interaction and adaptive thermogenesis are thyroid hormone receptor isoform--specific.

In newborns and small mammals, cold-induced adaptive (or nonshivering) thermogenesis is produced primarily in brown adipose tissue (BAT). Heat production is stimulated by the sympathetic nervous system, but it has an absolute requirement for thyroid hormone. We used the thyroid hormone receptor-beta--selective (TR-beta--selective) ligand, GC-1, to determine by a pharmacological approach whether adaptive thermogenesis was TR isoform--specific. Hypothyroid mice were treated for 10 days with varying doses of T3 or GC-1. The level of uncoupling protein 1 (UCP1), the key thermogenic protein in BAT, was restored by either T3 or GC-1 treatment. However, whereas interscapular BAT in T3-treated mice showed a 3.0 degrees C elevation upon infusion of norepinephrine, indicating normal thermogenesis, the temperature did not increase (<0.5 degrees C) in GC-1--treated mice. When exposed to cold (4 degrees C), GC-1--treated mice also failed to maintain core body temperature and had reduced stimulation of BAT UCP1 mRNA, indicating impaired adrenergic responsiveness. Brown adipocytes isolated from hypothyroid mice replaced with T3, but not from those replaced with GC-1, had normal cAMP production in response to adrenergic stimulation in vitro. We conclude that two distinct thyroid-dependent pathways, stimulation of UCP1 and augmentation of adrenergic responsiveness, are mediated by different TR isoforms in the same tissue.

Acetates↗
PubMed2001

Differential regulation of the human sodium/iodide symporter gene promoter in papillary thyroid carcinoma cell lines and normal thyroid cells.

The absence of TSH-stimulated radioiodide uptake in differentiated thyroid cancer is associated with a high recurrence rate and reduced survival. We studied regulation of the sodium/iodide symporter gene in human papillary thyroid cancer cell lines (BHP) and primary human thyroid cells. BHP cells expressed very low levels of sodium/iodide symporter mRNA and did not concentrate iodide, but iodide uptake was restored to levels seen in FRTL-5 rat thyroid cells by stable transfection of a sodium/iodide symporter cDNA. Sodium/iodide symporter gene expression, therefore, was necessary and sufficient for iodide uptake in BHP cells. We cloned the human sodium/iodide symporter gene 5'-flanking region and analyzed progressive 5'-deletions in transient transfections. We identified a region, -596 to -268, essential to confer full promoter activity in primary normal human thyroid cells. Sodium/iodide symporter promoter activity in four BHP cell lines, however, was markedly reduced, consistent with down-regulation of the endogenous sodium/iodide symporter gene. Nuclear extracts from BHP 2-7 cells had reduced or absent binding to regions of the sodium/iodide symporter promoter shown to be critical for expression, compared with nuclear extracts from FRTL-5 cells. Competition studies indicated that these nuclear proteins were not known thyroid transcription factors. Modifications of the sodium/iodide symporter promoter with demethylation or histone acetylation did not increase sodium/iodide symporter expression, and no deletions of the critical regulatory region were identified in the endogenous gene in BHP cells. Regulation of the sodium/iodide symporter 5'-flanking region in transient transfection paralleled endogenous sodium/iodide symporter expression. Reduced expression of potential novel nuclear factor(s) in these cell lines may contribute to reduced sodium/iodide symporter expression resulting in absence of iodide uptake in some papillary thyroid cancers.

Base Sequence↗
PubMed2001

Thyroid hormone stimulation of osteocalcin gene expression in ROS 17/2.8 cells is mediated by transcriptional and post-transcriptional mechanisms.

We investigated the mechanism of thyroid hormone regulation of osteocalcin (OC) gene expression in osteoblast-like cells (ROS 17/2.8). Treatment with tri-iodothyronine (T3) (10(-8) M) increased OC mRNA levels by approximately 3-fold after 24 h and reached a maximum, approximately 5.4-fold, after 48 h. The mRNA levels of other bone-specific genes, alkaline phosphatase and osteopontin, were not affected by T3 treatment. Interestingly, T3 induction of OC mRNA varied according to cell density: approximately 4-fold at approximately 1x10(5) cells/dish and 1.5-fold at 40-60x10(5) cells/dish. The magnitude of OC mRNA induction by T3 was approximately 40% lower than induction by 1,25 dihydroxyvitamin D3 (1,25D3) alone, and the combination of T3+1,25D3 did not further stimulate OC mRNA levels. T3 induction of OC mRNA was not affected by treatment with cycloheximide (10 microg/ml) for 5 h indicating that new protein synthesis is not required for the response. To study the half-life of OC mRNA, ROS 17/2.8 cells were incubated with actinomycin D. The basal half-life of OC mRNA (means+/-s.e.m.) was 6.4+/-0.2 h which was increased significantly with either T3 or 1,25D3 treatment to 10.9+/-0.6 h and 13.5+/-0.4 h respectively. T3 modestly up-regulated the rate of OC gene transcription (1.7+/-0.2-fold) as determined by run-off assay. T3 did not induce a reporter construct containing the rat OC gene (rOC) 5'-flanking region (to -1750 bp) or the previously described rOC vitamin D response element, when transfected into ROS 17/2.8 cells. In conclusion, T3 up-regulates the OC mRNA expression in ROS 17/2.8 cells in a dose-, time- and cell confluence-dependent fashion, and does so by transcriptional and post-transcriptional mechanisms. The greater T3 induction of OC expression in ROS 17/2.8 cells at low cell density is consistent with findings of thyroid hormone action on bone development.

Animals↗
PubMed2000

Retinoic acid induces sodium/iodide symporter gene expression and radioiodide uptake in the MCF-7 breast cancer cell line.

The sodium/iodide symporter (NIS) stimulates iodide uptake in normal lactating breast, but is not known to be active in nonlactating breast or breast cancer. We studied NIS gene regulation and iodide uptake in MCF-7 cells, an estrogen receptor (ER)-positive human breast cancer cell line. All-trans retinoic acid (tRA) treatment stimulated iodide uptake in a time- and dose-dependent fashion up to approximately 9.4-fold above baseline. Stimulation with selective retinoid compounds indicated that the induction of iodide uptake was mediated by retinoic acid receptor. Treatment with tRA markedly stimulated NIS mRNA and immunoreactive protein ( approximately 68 kDa). tRA stimulated NIS gene transcription approximately 4-fold, as shown by nuclear run-on assay. No induction of iodide uptake was observed with RA treatment of an ER-negative human breast cancer cell line, MDA-MB 231, or a normal human breast cell line, MCF-12A. The iodide efflux rate of tRA-treated MCF-7 cells was slow (t(1/2) = 24 min), compared with that in FRTL-5 thyroid cells (t(1/2) = 3.9 min), favoring iodide retention in MCF-7 cells. An in vitro clonogenic assay demonstrated selective cytotoxicity with (131)I after tRA stimulation of MCF-7 cells. tRA up-regulates NIS gene expression and iodide uptake in an ER-positive breast cancer cell line. Stimulation of radioiodide uptake after systemic retinoid treatment may be useful for diagnosis and treatment of some differentiated breast cancers.

Alitretinoin↗
PubMed2000

Tissue-specific actions of thyroid hormone: insights from animal models.

The major developmental targets for thyroid hormone are the brain, small intestine, and bone. Clear defects in gene regulation and tissue function as a consequence of TR gene inactivation can additionally be shown in the pituitary, hypothalamus, heart, and liver. TR gene knockout models show a clear distinction between thyroid hormone requirements for development and those that are required for functions in the adult animal. T3-mediated gene repression appears especially important in a number of tissues including brain, pituitary, and the heart. Preliminary evaluation of the combined TR knockout models suggests that hypothyroidism is associated with more significant abnormalities than receptor deficiency, indicating that the repressive action of the unliganded receptor may have physiological relevance. These various animal models should be very useful to design and test thyroid hormone analogues to selectively stimulate desired thyroid hormone actions.

Animals↗
PubMed2000

Interactions between the prohormone convertase 2 promoter and the thyroid hormone receptor.

The majority of prohormones are cleaved at paired basic residues to generate bioactive hormones by prohormone convertases (PCs). As PC1 and PC2, two neuroendocrine-specific PCs, appear to be the key enzymes capable of processing a variety of prohormones, alterations of PC2 and/or PC1 levels will probably have a profound effect on hormonal homeostasis. We investigated the regulation of PC2 messenger RNA (mRNA) by thyroid hormone using GH3 cells to demonstrate that T3 negatively regulated PC2 mRNA levels in a dose- and time-dependent fashion. Functional analysis of progressive 5'-deletions of the human (h) PC2 promoter luciferase constructs in GH3 cells demonstrated that the regulation probably occurs at the transcriptional level, and that putative negative thyroid hormone response elements were located within the region from -44 to + 137 bp relative to the transcriptional start site. Transient transfections in JEG-3 cells and COS-1 cells showed that the suppressive effect of T3 was equally mediated by the thyroid hormone receptor (TR) isoforms TRalpha1 and TRbeta1. Electrophoretic mobility shift assays using purified TRal and retinoid X receptor-beta protein as well as GH3 nuclear extracts showed that regions from +51 to +71 bp and from +118 to +137 bp of the hPC2 promoter bind to TRalpha1 as both a monomer and a homodimer and with TRalpha1/retinoid X receptor-beta as a heterodimer. Finally, the in vivo regulation of pituitary PC2 mRNA by thyroid status was demonstrated in rats. These results demonstrate that T3 negatively regulates PC2 expression at the transcriptional level and that functional negative thyroid hormone response elements exist in the hPC2 promoter. We postulate that the alterations of PC2 activity may mediate some of the pathophysiological consequences of hypo- or hyperthyroidism.

Animals↗
PubMed2000

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.

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.

Carrier Proteins↗
PubMed1999

Maternal hypothyroidism: recognition and management.

Women with compensated early thyroid failure, or those from areas of reduced iodine intake, may first be found to be hypothyroid during pregnancy. In women with previously diagnosed hypothyroidism already on thyroxine (T4) replacement therapy, pregnancy is often associated with an increased dose requirement. The mechanism producing this increased requirement is not known, but it is likely to be the result of a number of factors that may differ depending on the stage of pregnancy. An increased T4 dose requirement is typically seen by the first trimester, can continue to increase throughout pregnancy, and reverts to the prepregnancy dose requirement after delivery. The magnitude of the increased T4 requirement is related to the etiology of hypothyroidism. Monitoring thyroid status and adjusting the T4 dose during pregnancy is a challenge due to changes in T4 metabolism throughout pregnancy.

Female↗
PubMed1998

Mechanisms of thyroid hormone action. Implications for the clinical manifestation of thyrotoxicosis.

Serum thyroid hormone concentrations alone do not explain the variability and severity of the range of symptoms observed in thyrotoxic patients. Despite gaps in our understanding of the links between the clinical manifestations of thyrotoxicosis and the underlying mechanisms, much has been learned. A limited number of markers directly reflect T3 action. The future elucidation of T3 targets that mediate these effects should ultimately lead to additional clinical markers of tissue-specific T3 action. The availability of such tests should allow for more specific treatment of individual patients.

Amino Acid Sequence↗
PubMed1997

Maternal thyroid function: interpretation of thyroid function tests in pregnancy.

Thyroid status is frequently assessed during pregnancy, both to evaluate suspected thyroid abnormalities, and to monitor the status, of pre-existing thyroid disease. However, the production, circulation, and disposal of thyroid hormone are all altered in pregnancy. Interpretation of thyroid function studies in the pregnant patient must be performed based on an understanding of the normal physiologic changes at each stage of pregnancy. Examples of pregnancy-associated changes include, estrogen-stimulated increase in serum thyroxine binding globulin, chorionic gonadotropin stimulated T4 and T3 production from the thyroid gland, and accelerated degradation of thyroid hormone by the placenta. The serum-TSH alone is usually not adequate to assess thyroid status in pregnancy, and the various conditions that can effect this measurement are described. The pattern of thyroid studies in pregnancy is important in diagnosing thyroid disease, and it may indicate physiologic adaptations to optimize maternal thyroid status for fetal development.

Female↗
PubMed1996

Characterization of the promoter of the rat sarcoplasmic endoplasmic reticulum Ca2+-ATPase 1 gene and analysis of thyroid hormone responsiveness.

Relaxation of skeletal muscle requires the re-uptake of Ca2+, which is mediated by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Thyroid hormone (T3) stimulates the expression of the SERCA1 isoform, which is essential for fast skeletal muscle fiber phenotype. We have cloned and studied the first 962 base pairs of the 5'-flanking region of the rat SERCA1 gene. This sequence was tested for T3-regulated expression in transient transfection experiments using COS7 cells and for binding of thyroid hormone receptor (TR) alpha in mobility shift assays. A construct of the 5'-flanking region and a reporter gene was unresponsive to T3 in the absence of co-transfected thyroid hormone receptor. In the presence of TRalpha, a T3 induction ratio of almost 4.0 was found, and this induction ratio was doubled with co-transfection of an RXR expression plasmid. Analysis of progressive 5'-deletion fragments of the sequence indicated multiple regions involved in T3 responsiveness. Three regions, R1, R2, and R3, were identified that bound TR complexes in mobility shift assays and conferred T3 responsiveness to a heterologous promoter. The most potent of these thyroid hormone response elements, R3, increased the 2-fold background T3 stimulation of the thymidine kinase promoter to nearly 6-fold. Detailed analysis of this element showed that four TR-binding half-sites, comprising two independent thyroid hormone response elements, interact cooperatively to give the maximal T3 response. T3 regulation of SERCA1 expression is mediated by a complex thyroid hormone response element that may serve to provide a greater range of response in interaction with nuclear receptor partners or cell-specific transcription factors.

Animals↗
PubMed1996

Structural features of thyroid hormone response elements that increase susceptibility to inhibition by an RTH mutant thyroid hormone receptor.

The chicken lysozyme silencer F2 (F2) thyroid hormone response element (TRE) contains an unusual everted palindromic arrangement, has a high affinity for thyroid hormone receptor (TR) homodimers, and is especially sensitive to dominant negative inhibition by, the T3 resistance (RTH) mutant TR beta P453H. We used various TREs and TR mutations to determine the mechanisms for this sensitivity. Changing the F2 orientation from an everted palindrome to a direct repeat with a 4-bp gap (DR+4) (F2-DR) decreased the sensitivity to inhibition at high T3 concentrations, while a loss of this sensitivity occurred with a palindromic arrangement of these same half-sites. F2 contains the dinucleotide TG 5' to each consensus half-site conforming to the optimal TR-binding octamer, YRRGGTCA. A T to A change in position 1 of both F2 half-sites markedly reduced T3-induction, yet only slightly reduced TR homodimer or TR-retinoid X receptor (RXR) heterodimer binding. The TR beta ninth heptad mutation, L428R, prevents TR heterodimerization with RXR and eliminates the inhibitory effect of the P453H mutant TR on the F2-DR, but not the F2 element. Structural features of a TRE that favor strong TR binding of both TR homodimers and TR-RXR heterodimers containing the mutant TR, such as the everted palindromic conformation or the optimal TR-binding consensus octamer, enhance the sensitivity of a TRE to inhibition by the mutant TR. Thus, both half-site orientation and sequence contribute to the sensitivity of a given TRE to dominant negative inhibition by a mutant TR.

Amino Acid Sequence↗
PubMed1995

Prospective studies of thyroid function in patients receiving gold therapy.

Gold inhibits the Type I deiodinase that provides the bulk of circulating T3 in humans. We prospectively studied thyroid function in patients receiving increasing parenteral cumulative gold doses. Eight consecutive euthyroid patients with rheumatoid or psoriatic arthritis who were initiating intramuscular gold therapy were enrolled. Serum thyroid hormone levels (total T4, T3, and rT3) and TSH were measured for each subject at various levels during gold therapy. For analysis, the free T4 and free T3 indices, TSH concentrations, and T4/T3 ratios were correlated with cumulative gold dose. Neither individual nor pooled linear regressions showed a significant correlation between cumulative gold dose and any of the thyroid function parameters. Thyroid function is not affected in patients receiving up to 1500 mg of gold compounds. The most likely explanation for this is that gold principally accumulates in the Kupffer cells and renal cortex and these cells do not express Type I deiodinase.

Adult↗
PubMed1995

Retinoid-X receptor (RXR) differentially augments thyroid hormone response in cell lines as a function of the response element and endogenous RXR content.

Retinoid-X receptor (RXR) forms heterodimers with thyroid hormone receptor (TR) and significantly enhances binding to thyroid hormone response elements (TREs). Expression of RXR in a transient transfection assay augments the T3 response, but the influences of the specific cell line and TRE used have not been systematically studied. We determined RXR alpha and -beta augmentation of the TR alpha-mediated T3 response in transient transfection assays of COS, JEG, and mouse embryonic stem (ES) cell lines for a series of eight wild-type thyroid hormone (T3) and retinoic acid response elements (previously shown to bind TR). RXR augmented T3-induced expression in COS and ES cells (1.5- to 4-fold greater expression with added RXR compared to TR alone), but had minimal effect on augmentation of response in JEG cells. For most elements studied there was a proportional augmentation of basal and T3-stimulated expression. TREs from rat GH and laminin-B1, however, had relatively higher levels of T3-induced expression as a result of RXR cotransfection (T3 induction ratios increased 2-fold or greater). Previous characterization of these elements demonstrates that they contain more than two hexameric binding domains, all of which can simultaneously bind TR. The influence of endogenous RXR expression in a cell line on RXR augmentation of the T3 response was determined. RXR alpha and -beta messenger RNA (mRNA) expression was quantitated by Northern blot in each cell line. COS and JEG cells expressed almost exclusively RXR alpha mRNA, although expression was almost 2-fold higher in JEG compared to COS cells (12 +/- 2.5 vs. 6.8 +/- 0.5 density units relative to actin; mean +/- SE; P < 0.05). ES cells expressed only RXR beta mRNA, but at a very low level (0.4 +/- 0.1). Nuclear extracts prepared from JEG and COS cells augmented TR binding proportional to the endogenous RXR mRNA expression, and the heterodimer band was supershifted by the addition of antibody to RXR alpha. Nuclear extracts from ES cells had no detectable TR heterodimer binding to a range of response elements. RXR augmentation of the T3 response differs among cell lines and is greater in those with reduced endogenous RXR. Furthermore, the functional augmentation of the T3 response ratio by RXR is likely to require additional sequences contained in only a subset of elements in which RXR augments TR binding.

Animals↗
PubMed1994

The molecular basis of thyroid hormone action.

Progress has been made in understanding the molecular basis of a number of clinical manifestations of thyroid disease, yet many questions remain. Why are there two thyroid hormone-receptor genes? Is the function of each of the two receptors indeed unique? How T3 receptors interact with other nuclear proteins and DNA-binding sites and how these interactions are influenced by T3 is incompletely understood. The developmental regulatory role of T3 receptor alpha 1 and its non-T3-binding alpha 2 variant needs to be defined. Most T3-regulated processes, especially those related to metabolism, muscle contraction, and brain development, function in concert with a number of other regulatory factors. The therapeutic applications of knowledge gained about the basic mechanisms of thyroid hormone action should ultimately extend beyond thyroid disease to processes regulated or influenced by T3; these include cardiac function, lipid metabolism, pituitary hormone secretion, and neural development.

Amino Acid Sequence↗