Norman L. Eberhardt

Thyroid hormone receptors and responses

Publisher Summary This chapter discusses the responses of thyroid hormone receptors. The thyroid hormone receptor is unique in that it is an intrinsic chromosomal nonhistone protein known to be involved in the regulation of the expression of specific genes. These receptors are bound to chromatin in the absence or the presence of thyroid hormone. They can be solubilized from chromatin and can bind to DNA. The hormone (mainly T4 or T3) enters the cell where it can be metabolized. Several metabolites of thyroxine (T4), such as T3, 3, 3’-T2, reverse T3, triac, and tetrac, have biological and receptor-binding activity; however, quantitatively, T3 is the most important hormone in vivo. This model for thyroid hormone action contrasts with the case of steroid hormones. Whereas the chromatin stimulates the capability of the thyroid hormone–receptor to bind the hormone, steroids stimulate the binding of their receptors to chromatin. Shortly after the interaction of thyroid hormone with the receptor, there is a major increase in the chromatins capacity to bind bacterial RNA polymerase and the distribution of chromatin proteins is altered. These data suggest that the hormone rapidly modifies chromatin in some way. These influences nevertheless are transmitted into effects only on the expression of a small subset of the cellular genes.

Cooperative binding of TEF-1 to repeated GGAATG-related consensus elements with restricted spatial separation and orientation.

The human transcriptional enhancer factor (TEF) family includes TEF-1, TEF-3, TEF-4, and TEF-5. The TEFs share a highly conserved 68-amino acid TEA/ATTS DNA-binding domain, which binds to SV40 GT-IIC (GGAATG), SphI (AGTATG), SphII (AGCATG), and muscle-specific M-CAT (GGTATG) enhansons. We determined the optimal DNA-binding consensus sequence for TEF-1. Using a purified GST-TEF-1 fusion protein and a random pool of synthetic oligonucleotides, 31 independent clones were obtained after six rounds of binding site selection. DNA sequences analysis revealed that 16 clones contained direct repeats with a 3-bp spacer (DR3), and 15 clones contained a single binding site. The predominate consensus half-site was GGAATG (67%), and the other elements were of the form G(A)GA(T/C)ATG. The TEF-1 bound to the DR3 as a dimer in a cooperative manner. Cooperative binding was dependent on the spacing and orientation of the half-sites and was inhibited by deoxycholate treatment, providing evidence that protein-protein interactions were involved. The data suggest that TEF dimerization is important for its ability to modulate gene transcription.

Hormonal domains of response: actions of glucocorticoid and thyroid hormones in regulating pleiotropic responses in cultured cells.

Publisher Summary This chapter discusses the pleiotropic responses of thyroid and glucocorticoid hormones in cultured hepatoma cells (H35 and HTC) and cultured anterior pituitary cells (GH3). Both hormones influence a limited and highly specific domain in each cell type. Approximately 1% or less of the detected gene products are affected by each hormone. Evidence presents that there is only a very limited overlap in the glucocorticoid domains of HTC and H35 cells, as only tyrosine amino-transferase is induced by dexamethasone in both cell types. There can be heterogenity in glucocorticoid domains among parenchymal cells from livers of the same strain of rats. Alternatively, differences in the levels of expression of domain members or transformation-dependent influences on the glucocorticoid domains of different cell lines can account for these results. In addition, the glucocorticoid hormone domains between GH3 and either HTC or H35 cells are almost completely non-identical. Overlap between the glucocorticoid and thyroid hormone domains has been demonstrated in GH3 cells. The two hormones can regulate the same gene product in one of three ways: conduction, co-repression, or induction by one hormone and repression by the other. Both glucocorticoid and thyroid hormones are capable of inducing and repressing individual domain members.

RNA Sequencing Identifies Multiple Fusion Transcripts, Differentially Expressed Genes, and Reduced Expression of Immune Function Genes in BRAF (V600E) Mutant vs BRAF Wild-Type Papillary Thyroid Carcinoma

CONTEXT The BRAF V600E mutation (BRAF-MUT) confers an aggressive phenotype in papillary thyroid carcinoma, but unidentified additional genomic abnormalities may be required for full phenotypic expression. OBJECTIVE RNA sequencing (RNA-Seq) was performed to identify genes differentially expressed between BRAF-MUT and BRAF wild-type (BRAF-WT) tumors and to correlate changes to patient clinical status. DESIGN BRAF-MUT and BRAF-WT tumors were identified in patients with T1N0 and T2-3N1 tumors evaluated in a referral medical center. Gene expression levels were determined (RNA-Seq) and fusion transcripts were detected. Multiplexed capture/detection and digital counting of mRNA transcripts (nCounter, NanoString Technologies) validated RNA-Seq data for immune system-related genes. PATIENTS BRAF-MUT patients included nine women, three men; nine were TNM stage I and three were stage III. Three (25%) had tumor infiltrating lymphocytes. BRAF-WT included five women, three men; all were stage I, and five (62.5%) had tumor infiltrating lymphocytes. RESULTS RNA-Seq identified 560 of 13 085 genes differentially expressed between BRAF-MUT and BRAF-WT tumors. Approximately 10% of these genes were related to MetaCore immune function pathways; 51 were underexpressed in BRAF-MUT tumors, whereas 4 (HLAG, CXCL14, TIMP1, IL1RAP) were overexpressed. The four most differentially overexpressed immune genes in BRAF-WT tumors (IL1B; CCL19; CCL21; CXCR4) correlated with lymphocyte infiltration. nCounter confirmed the RNA-Seq expression level data. Eleven different high-confidence fusion transcripts were detected (four interchromosomal; seven intrachromosomal) in 13 of 20 tumors. All in-frame fusions were validated by RT-PCR. CONCLUSION BRAF-MUT papillary thyroid cancers have reduced expression of immune/inflammatory response genes compared with BRAF-WT tumors and correlate with lymphocyte infiltration. In contrast, HLA-G and CXCL14 are overexpressed in BRAF-MUT tumors. Sixty-five percent of tumors had between one and three fusion transcripts. Functional studies will be required to determine the potential role of these newly identified genomic abnormalities in contributing to the aggressiveness of BRAF-MUT and BRAF-WT tumors.

FLUORESCENT RHODAMINE-LABELED THYROID HORMONE DERIVATIVES Synthesis and binding to the thyroid hormone nuclear receptor

Studies of the transport, intracellular distribution and interaction of thyroid hormones with specific subcellular components have depended upon the use of radiolabeled hormones. Application of this methodology to the study of transport in intact cells, however, is limited because of the difficulties in differentiating transported hormone from bound hormone at the cell surface. Therefore, it is important to devise alternate techniques for studying the trans- port of thyroid hormones in intact cells. A highly sensitive technique termed “video intensification microscopy’has been used [l-3] to study the binding and ~te~~ation of fluorescence-labeled peptide hormones and proteins in living cells. To apply this technique to thyroid hormones we have synthesized rhodamine-labeled derivations of iodothyronines directly to visualize their uptake, transport and distribution in cells. To determine if the derivatives retained biolo~c~ specificity, we studied their interaction with the thyroid hormone nuclear receptor [7]. A growing body of evidence indicates that many responses of thyroid hormones may be explained by their interac- tion with a chromatin-localized receptor which sub- sequently influences the expression of specific genes

Clustering of the B Cell Receptor Is Not Required for the Apoptotic Response

We examined the role of BCR cell membrane redistribution in anti-IgM-induced apoptosis in three human B cell lines, RA#1, 2G6, and MC116, that differ in their relative levels of sIgM expression. The apoptotic response was found to be dependent on the nature of the anti-IgM and the cell line. In the cell lines, RA#1 and MC116, sIgM aggregated into patches that were insensitive to the disruption of cholesterol-rich membrane microdomains by nystatin or beta-MCD. The B cell line 2G6 was able to reorganize sIgM into a tight coalescent cap upon anti-IgM treatment. However, in this case, the lipid raft inhibitors nystatin and beta-MCD disrupted the patching. In 2G6 cells, BCR-mediated apoptosis was not affected by nystatin treatment, whereas it increased in beta-MCD pretreated cells. Thus, no evident correlation was found between apoptosis and BCR cell membrane redistribution or lipid raft formation in either of the three cell lines. The data indicate that the apoptotic signal transduction pathway is independent of BCR translocation into lipid rafts and/or aggregation.

Kinetics of the apoptotic response induced by anti-IgM engagement of the B cell receptor is dependent on the density of cell surface immunoglobulin M expression.

The effect of B cell receptor (BCR) density on anti-BCR-induced apoptosis was assessed in Ramos cell lines, expressing low, medium, or high levels of surface IgM (sIgM(LO), sIgM(MED), sIgM(HI)). All cells required a 6-mug/ml threshold of anti-IgM to elicit apoptosis. Anti-IgM treatment of sIgM(LO) cells induced growth inhibition and limited dose-independent apoptosis. Anti-IgM treatment of sIgM(MED) cells induced dose-independent death with a 32-h lag. Ligation of the BCR in the sIgM(HI) cells induced rapid apoptosis beginning by 6 h, which was dose-dependent. Secondary crosslinking reagents did not affect apoptosis, and this effect was independent of anti-IgM concentration, time, or sIgM density. These results suggest that the response to BCR engagement strongly depends on the cell surface receptor density.

Development of a Multidisciplinary, Multicampus Subspecialty Practice in Endocrine Cancers

OBJECTIVES Relative to more abundant neoplasms, endocrine cancers have been historically neglected, yet their incidence is increasing. We therefore sought to build interest in endocrine cancers, improve physician experience, and develop innovative approaches to treating patients with these neoplasms. METHODS Between 2005 and 2010, we developed a multidisciplinary Endocrine Malignancies Disease Oriented Group involving all 3 Mayo Clinic campuses (Rochester, Minnesota; Jacksonville, Florida; and Scottsdale, Arizona). In response to higher demand at the Rochester campus, we sought to develop a Subspecialty Tumor Group and an Endocrine Malignancies Tumor Clinic within the Division of Medical Oncology. RESULTS The intended groups were successfully formed. We experienced difficulty in integration of the Mayo Scottsdale campus resulting from local uncertainty as to whether patient volumes would be sufficient to sustain the effort at that campus and difficulty in developing enthusiasm among clinicians otherwise engaged in a busy clinical practice. But these obstacles were ultimately overcome. In addition, with respect to the newly formed medical oncology subspecialty endocrine malignancies group, appointment volumes quadrupled within the first year and increased 7 times within 2 years. The number of active therapeutic endocrine malignancies clinical trials also increased from 1 in 2005 to 5 in 2009, with all 3 Mayo campuses participating. CONCLUSIONS The development of subspecialty tumor groups for uncommon malignancies represents an effective approach to building experience, increasing patient volumes and referrals, and fostering development of increased therapeutic options and clinical trials for patients afflicted with otherwise historically neglected cancers.PURPOSE Relative to more abundant neoplasms, endocrine cancers have been historically neglected, yet their incidence is increasing. We therefore sought to build interest in endocrine cancers, improve physician experience, and develop innovative approaches to treating patients with these neoplasms. METHODS Between 2005 and 2010, we developed a multidisciplinary Endocrine Malignancies Disease Oriented Group involving all three Mayo Clinic campuses (Rochester, MN; Jacksonville, FL; and Scottsdale, AZ). In response to higher demand at the Rochester campus, we sought to develop a Subspecialty Tumor Group and an Endocrine Malignancies Tumor Clinic within the Division of Medical Oncology. RESULTS The intended groups were successfully formed. We experienced difficulty in integration of the Mayo Scottsdale campus resulting from local uncertainty as to whether patient volumes would be sufficient to sustain the effort at that campus and difficulty in developing enthusiasm among clinicians otherwise engaged in a busy clinical practice. But these obstacles were ultimately overcome. In addition, with respect to the newly formed medical oncology subspecialty endocrine malignancies group, appointment volumes quadrupled within the first year and increased seven times within two years. The number of active therapeutic endocrine malignancies clinical trials also increased from one in 2005 to five in 2009, with all three Mayo campuses participating. CONCLUSION The development of subspecialty tumor groups for uncommon malignancies represents an effective approach to building experience, increasing patient volumes and referrals, and fostering development of increased therapeutic options and clinical trials for patients afflicted with otherwise historically neglected cancers.

Cytogenetic Abnormalities Associated with Endocrine Neoplasia

During the past decade, cytogenetic studies of solid human neoplasms have come of age. Although technically more difficult and often ambiguous in comparison with cytogenetic studies of leukemias and lymphomas (1) cytogenetic studies of many solid tumors have yielded valuable insights into tumor pathogenesis and prognosis. In addition, in the last decade we have seen increasing application of molecular genetic techniques to the study of cancer, which has contributed greatly to our understanding of mechanisms controlling cell growth. Specific genetic alterations responsible for the pathogenesis of a number of neoplasms have been identified.