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N L Eberhardt

Publications and source records attributed to N L Eberhardt.

At least 37 records · Page 2Linked to original sources
PubMed1997

Frequent loss of heterozygosity on chromosomes 3p and 17p without VHL or p53 mutations suggests involvement of unidentified tumor suppressor genes in follicular thyroid carcinoma.

Follicular thyroid carcinoma (FTC) exhibits frequent loss of heterozygosity (LOH) on chromosomes 10q and 3p, suggesting involvement of tumor suppressor genes. We screened 14 FTC (10 Hurthle cell carcinomas and 4 nonoxyphilic FTC), 14 papillary thyroid carcinomas, and 7 follicular adenomas for LOH on chromosome arms 1p, 3p, 3q, 10p, 10q, 11p, 11q, 13q, 17p, and 17q. LOH was more frequent in FTC than in follicular adenoma or papillary thyroid carcinoma. In FTC, rates of LOH on 3p (86%), 17p (72%), and 10q (57%) were higher than the average rate of LOH (33%; P < 0.05). Most frequently involved were 3p21-25 and 17p13.1-13.3, the sites for the VHL (3p25-26) and p53 (17p13.1) tumor suppressors. We, therefore, characterized these genes by dideoxy fingerprinting and DNA sequencing. Two FTC had mutations in p53, but only 1 of these exhibited LOH at 17p. No VHL gene mutations were found. Thus, neither p53 nor VHL genes play a significant role in the pathogenesis of differentiated thyroid cancer. LOH on 17p, but not on 3p or 10q, was correlated with mortality. Accordingly, 3p and 10q LOH may represent early, and 17p LOH late, events in FTC development. The data suggest the presence of novel tumor suppressor genes on chromosomes 3p and 17p that may be important in the pathogenesis of FTC.

Adenocarcinoma, Follicular↗
PubMed1997

Human chorionic somatomammotropin enhancer function is mediated by cooperative binding of TEF-1 and CSEF-1 to multiple, low-affinity binding sites.

The human chorionic somatomammotropin gene enhancer (CSEn) is composed of multiple enhansons (Enh) that share sequence similarities with those of the simian virus, SV40 enhancer (SVEn). The sequence homology includes two GT-IIC-like (Enh1 and Enh4) and three SphI/II-like enhansons (Enh2, Enh3, and Enh5). We previously showed that transcription enhancer factor 1 (TEF-1) and a 30-kDa placental-specific factor, chorionic somatomammotropin enhancer factor 1 (CSEF-1), bind to Enh4, which plays an essential role in enhancer function. In this study, we demonstrate that TEF-1 and CSEF-1 bind specifically to all the other GT-IIC- and SphI/II-like elements within CSEn with a broad range of binding affinities that vary between 0.005 and 0.15 that of Enh4. Each individual concatenated enhanson was able to stimulate hCS promoter activity in an orientation-independent manner in choriocarcinoma cells (BeWo) with an observed stimulation that was directly proportional to its relative binding affinity for TEF-1 and CSEF-1. These results indicate that CSEn function results from the cooperative interaction of TEF-1 and/or CSEF-1 binding to multiple, low-affinity GT-IIC- and SphI/II-like enhansons within the enhancer.

Binding Sites↗
PubMed1997

The human chorionic somatomammotropin enhancers form a composite silencer in pituitary cells in vitro.

The human GH (GH) gene family includes the pituitary-specific hGH-1, placental-specific chorionic somatomammotropin (hCS-5, hCS-2, and hCS-1), and hGH-2 genes. These duplicated, nearly identical genes are localized on approximately 50 kb of DNA on chromosome 17q23-q24. An enhancer (CSEn2), located downstream of the hCS-2 gene, participates in mediating placental-specific hCS gene expression. In the preceding paper we demonstrated that CSEn2 activity derives from the cooperative binding of transcription factor-1, TEF-1, and a placental-specific factor CSEF-1 to multiple enhansons, Enh1-Enh5, that are related to the SV40 GT-IIC and SphI/SphII enhansons. Here we demonstrate that two copies of CSEn2 or a single copy of CSEn2 linked to either of the other two enhancers in the hGH/hCS locus, CSEn1 and CSEn5, act cooperatively to enhance hCS promoter activity in choriocarcinoma (BeWo) cells, but silence the promoter in pituitary GC cells. Mutation of Enh4, an essential GT-IIC-like enhanson in the context of the intact enhancer, abolishes silencer activity, and multimerized GT-IIC enhansons mimic the intact CSEn enhancer/silencer activities in BeWo and GC cells, respectively. By antibody-mediated supershift, Western, and far Western analyses, we identified TEF-1 as the GT-IIC-binding factor in pituitary cells. The data suggest that TEF-1 may be involved in pituitary-specific repression of placental GH/CS gene transcription through long-range interactions between the multiple CS enhancers present on the GH/CS gene locus.

Animals↗
PubMed1996

An efficient method for generation and subcloning of tandemly repeated DNA sequences with defined length, orientation and spacing.

Tandemly repeated DNA sequences generated from single synthetic oligonucleotide monomers are useful for many purposes. With conventional ligation procedures low yields and random orientation of oligomers makes cloning of defined repeated sequences difficult. We solved these problems using 2 bp overhangs to direct orientation and random incorporation of linkers containing restriction sites during ligation. Ligation products are amplified by PCR using the linker oligonucleotides as primers. Restriction digestion of the PCR products generate multimer distributions whose length is controlled by the monomer/linker ratio. The concatenated DNA fragments of defined length, orientation and spacing can be directly used for subcloning or other applications without further treatment.

Base Sequence↗
PubMed1996

TEF-1 transrepression in BeWo cells is mediated through interactions with the TATA-binding protein, TBP.

Transcription enhancer factor-1 (TEF-1) has been implicated in transactivating a placental enhancer (CSEn) that regulates human chorionic somatomammotropin (hCS) gene activity. We demonstrated that TEF-1 represses hCS promoter activity in choriocarcinoma (BeWo) cells (Jiang, S.W., and Eberhardt, N.L. (1995) J. Biol. Chem. 270, 13609-13915), suggesting that TEF-1 interacts with basal transcription factors. Here we demonstrate that hTEF-1 overexpression inhibits minimal hCS promoters containing TATA and/or initiator elements, Rous sarcoma virus and thymidine kinase promoters in BeWo cells. Cotransfection of TEF-1 antisense oligonucleotides alleviated exogenous TEF-1-mediated repression and increased basal hCS promoter activity, indicating that endogenous TEF-1 exerts repressor activity. GST-TEF-1 fusion peptides fixed to glutathione-Sepharose beads retained in vitro-generated human TATA-binding protein, hTBP. The TEF-1 proline-rich domain was essential for TBP binding, but polypeptides also containing the zinc finger domain bound TBP with higher apparent affinity. TBP supershifted hTEF-GT-IIC DNA complexes, but TEF-1 inhibited in vitro binding of TBP to the TATA motif. Coexpression of TBP and TEF-1 in BeWo cells alleviated TEF-1-mediated transrepression, indicating that the TBP-TEF-1 interaction is functional in vivo. The data indicate that TEF-1 transrepression is mediated by direct interactions with TBP, possibly by inhibiting preinitiation complex formation.

Animals↗
PubMed1995

Involvement of a protein distinct from transcription enhancer factor-1 (TEF-1) in mediating human chorionic somatomammotropin gene enhancer function through the GT-IIC enhanson in choriocarcinoma and COS cells.

Previous studies suggested that transcription enhancer factor-1 (TEF-1) was involved in mediating the human chorionic somatomammotropin (hCS) gene enhancer (CSEn) function (Jiang, S.-W., and Eberhardt, N. L. (1994) J. Biol. Chem. 269, 10384-10392). We now show that an unrelated protein (CSEF-1) found in BeWo and COS-1 cells binds to the GT-IIC enhanson in CSEn and is correlated with CSEn activity in these cells. TEF-1 and CSEF-1 were distinguished by differential migration as GT-IIC complexes, thermal stability, molecular mass, and cross-reactivity with chicken TEF-1 antibodies. TEF-1 and CSEF-1 bound to the GT-IIC and Sph-I/Sph-II enhansons with identical binding properties, and in vitro generated TEF-1 competed with CSEF-1 binding to the GT-IIC motif, suggesting that their actions might be mutually exclusive. Up- and down-regulation of TEF-1 levels by expression systems and antisense oligonucleotides demonstrated that TEF-1 inhibited the hCS promoter in a manner independent of the enhancer or a known TEF-1 DNA binding site. The data suggest that TEF-1 may provide a counter-regulatory stimulus to the actions of CSEF-1, which may be involved in mediating enhancer stimulatory activity.

Base Sequence↗
PubMed1995

An initiator element is required for maximal human chorionic somatomammotropin gene promoter and enhancer function.

Previous studies have indicated that cell-specific expression of the human chorionic somatomammotropin (hCS) gene may be mediated by a placental-specific enhancer (CSEn). In the current studies, we have analyzed the promoter elements that are required for enhancer and promoter function in choriocarcinoma cells (BeWo). Mutation of both hCS GHF1 sites had no effect on promoter or enhancer activity. In contrast, mutation of the Sp1 site diminished basal and CSEn-stimulated transcription by approximately 75% and approximately 56%, respectively, indicating that Sp1 was necessary but not sufficient for maximal basal and enhancer-mediated transcription. Deletion and site-specific mutation of the proximal promoter region indicated that the TATA box and an initiator site (InrE) located between nucleotides -15/+1 of the hCS promoter were required for maximal promoter and enhancer function. Mutations of the InrE were associated with reduced basal and enhancer-stimulated activities and altered transcription initiation sites. A protein of 70-kDa mass, that was preferentially expressed in human choriocarcinoma cells (BeWo and JEG-3), bound specifically to the InrE. The data suggest that an initiator present in high concentrations in placental cells contributes to the control of cell-specific hCS gene expression at the promoter level and is required for maximal enhancer function.

Base Sequence↗
PubMed1995

Human islet amyloid polypeptide expression in COS-1 cells. A model of intracellular amyloidogenesis.

Non-insulin-dependent diabetes mellitus is characterized by concurrent loss of beta-cells and deposition of islet amyloid derived from islet amyloid polypeptide (IAPP). We have previously demonstrated that IAPP-derived amyloid forms intracellularly in humans with chronic excess insulin expression (eg, insulinoma and insulin receptor antibody-induced insulin resistance). To determine whether overexpression of IAPP results in intracellular amyloid in mammalian cells, we transfected COS cells with vectors expressing amyloidogenic human IAPP or non-amyloidogenic rat IAPP. Transfected COS-1 cells secreted comparable amounts of human IAPP and rat IAPP (2.1 to 2.8 nmol/L/48 hours). After 96 hours, 90% of cells expressing human IAPP contained amyloid fibrils and were degenerating or dead, whereas cells transfected with rat IAPP lacked amyloid and were viable. Thus, overexpression of human IAPP can result in intracellular amyloid formation that is associated with cell death, suggesting that intracellular amyloid may play a role in beta-cell loss in non-insulin-dependent diabetes mellitus.

Amyloid↗
PubMed1994

The human chorionic somatomammotropin gene enhancer is composed of multiple DNA elements that are homologous to several SV40 enhansons.

Previous studies indicate that a human chorionic somatomammotropin (hCS) gene enhancer (CSEn) associated with the growth hormone (hGH) gene locus is involved in directing cell-specific expression of the hCS genes in placenta. In the current studies, we report a detailed structural analysis of this enhancer. CSEn stimulated transcription of a variety of promoters, including the hCS, human growth hormone, thymidine kinase, and Rous sarcoma virus promoters, in human choriocarcinoma cell lines (BeWo and JEG-3) but not HeLa cells or rat somatolactotrophes (GC). Maximal enhancer activity was confined to a 242-base pair DNA segment. Of several CSEn subfragments, only the En 57/242 subfragment retained activity (33.5% wild-type). The CSEn DNA sequence contained direct and inverted repeat motifs and sequences related to the SV40 enhansons, GT-IIC, GT-I, and SphI/SphII. DNase I footprint analysis revealed that most of these sites were protected by nuclear proteins derived from BeWo, JEG-3, HeLa, and GC cells. Site-specific block mutation of the GT-IIC-related and inverted repeat motifs virtually abolished enhancer activity, and mutation of all but the GT-I-related motif resulted in significant loss (30-60%) of activity. These data demonstrate that the CS enhancer is comprised of multiple elements related to SV40 enhansons that interact cooperatively to generate enhancer function.

Animals↗
PubMed1994

Two CGTCA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells.

We established the cis-acting elements which mediate cAMP responsiveness of the human growth hormone (hGH) gene in transiently transfected rat anterior pituitary tumor GC cells. Analysis of the intact hGH gene or hGH 5'-flanking DNA (5'-FR) coupled to the hGh cDNA or chloramphenicol acetyltransferase or luciferase genes, indicated that cAMP primarily stimulated hGH promoter activity. Cotransfection of a protein kinase A inhibitory protein cDNA demonstrated that the cAMP response was mediated by protein kinase A. Mutational analysis of the hGH promoter identified two core cAMP response element motifs (CGTCA) located at nucleotides -187/-183 (distal cAMP response element; dCRE) and -99/-95 (proximal cAMP response element; pCRE) and a pituitary-specific transcription factor (GHF1/Pit1) binding site at nucleotides -123/-112 (dGHF1) which were required for cAMP responsiveness. GHF1 was not a limiting factor, since overexpression of GHF1 in cotransfections increased basal but not forskolin induction levels. Gel shift analyses indicated that similar, ubiquitous, thermostable protein(s) specifically bound the pCRE and dCRE motifs. The CGTCA motif-binding factors were cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1)-related, since the DNA-protein complex was competed by unlabeled CREB consensus oligonucleotide, specifically supershifted by antisera to CREB and ATF-1 but not ATF-2, and was bound by purified CREB with the same relative binding affinity (pCRE < dCRE < CREB) and mobility as the GC nuclear extract. UV cross-linking and Southwestern blot analyses revealed multiple DNA-protein interactions of which approximately 100- and approximately 45-kDa proteins were predominant; the approximately 45-kDa protein may represent CREB. These results indicate that CREB/ATF-1-related factors act coordinately with the cell-specific factor GHF1 to mediate cAMP-dependent regulation of hGH-1 gene transcription in anterior pituitary somatotrophs.

Animals↗
PubMed1994

Transforming growth factor-beta 1 induces growth inhibition of a human medullary thyroid carcinoma cell line despite an increase in steady state c-myc messenger ribonucleic acid levels.

Medullary thyroid cancer (MTC) is an endocrine tumor of the thyroid C-cells which provides an important experimental model for studies of tumor differentiation and progression. We investigated the effects of transforming growth factor-beta 1 (TGF beta 1) on the growth and functional characteristics of a human medullary thyroid carcinoma cell line (TT). Because the c-myc protooncogene may play an important role in the growth inhibition induced by TGF beta 1, we also assessed steady state c-myc messenger RNA (mRNA) levels in these cells. A 6-day exposure of TT cells to TGF beta 1 resulted in a dose-dependent inhibition of cell proliferation. In addition, TGF beta 1 exposure led to a 3-fold increase in nonadherent floating TT cells in the culture supernatants. The floating cells exhibited ultrastructural features of dying or apoptotic cells, including chromatin condensation, cytoplasmic and nuclear vesicularization, and DNA degradation with evidence of internucleosomal DNA "laddering." Despite inhibition of cell proliferation, steady state c-myc mRNA levels were 3.6 +/- 0.6-fold higher in cells exposed to TGF beta 1 compared to those in control cells (P < 0.001). Exposure of cells to a 15-base antisense c-myc oligonucleotide (10 microM) resulted in an attenuation of the TGF beta 1-induced growth inhibition and induction of cell death. TGF beta 1 also resulted in an approximately 3-fold decrease in steady state calcitonin and calcitonin gene-related peptide mRNA levels. Finally, using a sensitive bioassay for TGF beta, TT cells were shown to produce and activate significant amounts of TGF beta, particularly under conditions of serum deprivation. Our data thus indicate that TGF beta 1 has multiple effects on TT cell growth and function. It induces growth inhibition in the presence of an increase in steady state mRNA levels of the c-myc protooncogene, which is usually associated with cell proliferation. In addition, TGF beta 1 accelerates apoptosis in TT cells.

Base Sequence↗
PubMed1993

DNA bending by retinoid X receptor-containing retinoid and thyroid hormone receptor complexes.

Retinoid X receptors (RXR) have been identified as common subunits in the regulation of multiple hormonal signaling pathways. Using circular permutation and phasing analysis of specific response elements, we present evidence that RXR-retinoic acid receptor and RXR-thyroid hormone receptor heterodimer or RXR-RXR homodimer complexes induce directed DNA bends when bound to their cognate response elements. The extent of DNA bending induced by the RXR alpha-containing complexes varied and depended on the structure of the DNA-binding sites and the RXR partners. The overall bending orientation for RXR-containing complexes is directed toward the major groove of the DNA helix at the center of hormone response elements. Our observation implicates DNA bending as a possible mechanism underlying transcriptional regulation of distinct retinoid and thyroid hormone responsive genes.

Base Sequence↗
PubMed1993

Congenital gigantism due to growth hormone-releasing hormone excess and pituitary hyperplasia with adenomatous transformation.

The cause of gigantism in most patients is a GH-secreting pituitary tumor. In this report, a case of congenital gigantism due to probable central hypersection of GH-releasing hormone (GHRH) is described. Normal at birth (4.4 kg; 53 cm), our 7-yr-old male patient grew progressively thereafter to attain a height of 182 cm and a weight of 99.4 kg at the time of our evaluation. The markedly increased baseline plasma levels of GH (730 micrograms/L) did not suppress during a standard 3-h oral glucose tolerance test, but did increase 54% after iv infusion of GHRH. Baseline plasma levels of insulin-like growth factor-I, PRL, and immunoreactive GHRH were also markedly increased. Computed imaging of the head showed a large, partially cystic sellar and suprasellar mass. Extensive imaging studies did not localize a potential source of GHRH. Preoperative treatment with octreotide and bromocriptine for 4 months resulted in a 25% reduction of suprasellar tissue mass. The pituitary tissue removed at transsphenoidal and transfrontal operations showed massive somatotroph, lactotroph, and mammosomatotroph hyperplasia. Areas of GH- and PRL-secreting cell adenomatous transformation were also evident. No histological or immunohistochemical evidence of a pituitary source of GHRH was found. The peripheral plasma immunoreactive GHRH concentration remained unaffected by pharmacological and surgical interventions. We suspect that a congenital hypothalamic regulatory defect may be responsible for the GHRH excess in this case.

Adenoma↗
PubMed1993

Thyroid hormone resistance syndrome: correlation of dominant negative activity and location of mutations.

Generalized resistance to thyroid hormone (GRTH) is caused by multiple distinct mutations that cluster in two regions of the hormone-binding domain of the thyroid hormone beta-receptor. The mutant receptors are functionally inactive, but nevertheless inhibit normal receptor activity in a dominant negative manner. Four different GRTH mutants were studied in the transient expression assays to further examine their functional properties. The transcriptional activity of the mutant receptors correlated with their T3 binding affinities. Two distal region mutants with partial T3 binding were transcriptionally active at high T3 concentrations, but exhibited potent dominant negative activity at low T3 concentrations. Two proximal region mutants that did not bind to T3 were 5- to 10-fold less effective inhibitors of normal receptor function, indicating that dominant negative inhibition is not correlated with T3 binding activity. Each of the proximal and distal region mutants retain the ability to form heterodimers with accessory proteins and to bind to DNA effectively. Because the non-T3 binding thyroid hormone receptor isoform alpha 2 also exists in most tissues, its effects on mutant receptor function were also examined. The inhibitory activity of each of the GRTH mutants was potentiated by alpha 2 but only in the context of a positively regulated reporter gene. Thus, alpha 2 may selectively alter the degree of dominant negative activity that occurs for different target genes. We conclude that the locations of GRTH mutations may influence dominant negative activity by altering transactivating or other functions of the receptor, providing a potential basis for the phenotypic variability in different kindreds with GRTH.

Base Sequence↗
PubMed1993

Molecular mechanisms of thyroid hormone action.

This review summarizes our current understanding of the mechanisms of thyroid hormone action. Topics included are the structures of thyroid hormone response elements (TREs), thyroid hormone receptor (TR)-TRE interactions, and TR association with other proteins to illustrate their importance in modulating both positive and negative transcriptional activation of specific genes. Certain studies focusing on other members of the steroid/thyroid hormone receptor superfamily are covered to provide a current view of how this class of receptors stabilizes transcription initiation complexes.

Animals↗
PubMed1992

Negative thyroid hormone control of human growth hormone gene expression is mediated by 3'-untranslated/3'-flanking DNA.

The intact human growth hormone (hGH) gene is negatively regulated by triiodothyronine (T3) treatment in transfected rat pituitary tumor cells. We now demonstrate that this responsiveness is mediated by a negative thyroid hormone response element (nTRE) localized to the proximal 3'-untranslated/3'-flanking region (3'-UT/FR). This region binds thyroid hormone receptors specifically and with high affinity. nTRE function was promoter-dependent, since it suppressed the activity of a positive TRE in the human chorionic somatomammotropin promoter, partially repressed activity of the herpes simplex virus TK promoter, but did not function with the human actin or Rous sarcoma virus promoters. T3 treatment did not alter transcript termination sites nor selectively affect the stability of transcripts containing the hGH 3'-UT/FR when transcription was blocked by actinomycin D treatment. The function of the nTRE depended on its location in the 3'-UT/FR; it was inactive when positioned down-stream of the simian virus 40 (SV40) 3'-UT/FR, and it acted as a positive TRE when placed upstream of the hGH promoter. These results demonstrate a novel localization of a TRE with unique properties which suggests expanded mechanisms by which thyroid hormone receptors can affect gene expression.

Animals↗